Chimeric proteins

Chimeric proteins possessing Kunitz-type domain 1 of TFPI-2 and Kunitz-type domain 2 of TFPI are disclosed, as are muteins of TFPI and TFPI-2. Nucleic acid sequences, expression vectors and transformed host cells encoding and capable of producing the disclosed chimeric proteins and muteins are also disclosed. Finally, methods for prevention and treatment of septic shock using the chimeric proteins and muteins are disclosed.

BACKGROUND OF THE INVENTION 
This invention relates to chimeric proteins capable of simultaneously 
binding and inhibiting factor VIIa/tissue factor complex (factor VIIa/TF 
complex) and factor Xa, expression vectors coding for the proteins of the 
invention, host cells transformed with the expression vectors, methods for 
producing the proteins, pharmaceutical compositions containing the 
proteins, methods of treatment or prevention of septic shock using the 
proteins, methods of inhibiting coagulation disorders and monoclonal 
antibodies against the proteins. 
Human Tissue Factor Pathway Inhibitor (TFPI) is a plasma protease 
inhibitor. Based on homology study, TFPI appears to be a member of the 
Kunitz-type basic protease inhibitor gene superfamily. TFPI functions in 
at least two ways: 1) Inhibition of the catalytic activity of factor 
VIIa/TF complex and 2) By binding to the active site of factor Xa. The 
primary sequence of TFPI, deduced from its cDNA sequence, indicates that 
the protein contains three Kunitz-type domains. The first of these, 
Kunitz-type domain 1, is believed to be required for the efficient binding 
to and inhibition of factor VIIa/TF complex, which is enhanced by the 
presence of the second Kunitz-type domain, Kunitz-type domain 2. 
Kunitz-type domain 2 is required for the efficient binding to and 
inhibition of factor Xa by TFPI. The function of the third Kunitz-type 
domain, Kunitz-type domain 3, is unknown. TFPI has no known enzymatic 
activity and probably inhibits the activity of protease targets in a 
stoichiometric manner, namely, binding of one Kunitz-type domain to the 
active site of one protease molecule. TFPI is also known as Lipoprotein 
Associated Coagulation Inhibitor (LACI), tissue factor inhibitor (TFI) and 
extrinsic pathway inhibitor (EPI). 
Mature TFPI is a polypeptide of about 276 amino acids in length with a 
negatively charged amino terminal end and a positively charged carboxyl 
terminal end. The C-terminal tail (i.e., the sequence following the last 
cysteine residue of Kunitz-type domain 3) is highly basic and is believed 
to aid in the localization of TFPI to cell surfaces by binding to 
glycosaminoglycan (including heparin) or phospholipids found on cell 
surfaces. This cell surface localization property is believed to be 
important for full anticoagulant activity and for optimal inhibition of 
factor Xa TFPI contains 18 cysteine residues and forms 9 disulfide bridges 
when correctly folded. The primary sequence contains three Asn-X-Ser/Thr 
N-linked glycosylation consensus sites with asparagine residues at 
positions 145, 196, and 256. The carbohydrate component of mature TFPI is 
approximately 30% of the mass of the protein. Data from proteolytic 
mapping and mass spectral data imply that the native carbohydrate moieties 
are heterogeneous. Native TFPI is also found to be phosphorylated at the 
serine residue at position 2 of the protein to varying degrees. The role 
of phosphorylation at Ser-2 in TFPI function has yet to be elucidated. 
Recently, another protein with a high degree of structural and functional 
similarity to TFPI has been identified, as described in Sprecher et al. 
Proc. Natl. Acad Sci. U.S.A. 91:3353-3357 (1994). The predicted secondary 
structure of this 213 amino acid residue protein, called TFPI-2, is 
virtually identical to TFPI having three Kunitz-type domains, 9 disulfide 
bridges, an acidic amino terminus and a basic carboxy terminus. The three 
Kunitz-type domains of TFPI-2 exhibit 43%, 35% and 53% primary sequence 
identity with TFPI Kunitz-type domains 1, 2, and 3, respectively. Compared 
with TFPI, recombinant TFPI-2 strongly inhibits the amidolytic activity of 
factor VIIa/TF complex and weakly inhibits factor Xa activity. TFPI-2 is 
reported to bind with greater affinity to factor VIIa/TF complex than does 
TFPI, whereas TFPI binds to factor Xa with greater affinity than does 
TFPI-2. 
The presumed P1-reactive site in Kunitz-type domain 1 of TFPI-2 is 
arginine, as contrasted with lysine in TFPI. The P1-reactive site in 
Kunitz-domain 2 of TFPI-2 is glutamate, as contrasted with arginine in 
TFPI. Also, the Kunitz-type domain 2 of TFPI-2 contains two additional 
amino acid residues between the fourth and fifth cysteine residues. The 
spacer region between Kunitz-type domains 1 and 2 in TFPI-2 is much 
shorter than the corresponding TFPI spacer region. One or more of these 
differences may result in the different affinities of the two proteins for 
factor VIIa/TF complex and Xa. 
TFPI has been shown to prevent mortality in a lethal Escherichia coli (E. 
coli) septic shock baboon model. Creasey et al, J. Clin. Invest. 
91:2850-2860 (1993). Administration of TFPI at 6 mg/kg body weight shortly 
after infusion of a lethal dose of E. coli resulted in survival in all 
five TFPI-treated animals with significant improvement in quality of life, 
compared with a mean survival time for the five control animals of 39.9 
hours. The administration of TFPI also resulted in significant attenuation 
of the coagulation response, of various measures of cell injury and 
significant reduction in pathology normally observed in E. coli sepsis 
target organs, including kidneys, adrenal glands, and lungs. Due to its 
clot-inhibiting properties, TFPI may also be used to prevent problems 
associated with thrombosis and clotting such as during microvascular 
surgery. For example, U.S. Pat. No. 5,276,015 discloses the use of TFPI in 
a method for reducing thrombogenicity of microvascular anastomoses wherein 
TFPI is administered at the site of the microvascular anastomoses 
contemporaneously with microvascular reconstruction. 
TFPI has been isolated from human plasma and from human tissue culture 
cells, including HepG2, Chang liver and SK hepatoma cells. Recombinant 
TFPI has been expressed in mouse C, 127 cells, baby hamster kidney cells, 
Chinese hamster ovary cells and human SK hepatoma cells. Recombinant TFPI 
from the mouse C127 cells has been shown in animal models to inhibit 
tissue-factor induced coagulation. A non-glycosylated form of recombinant 
TFPI has also been produced and isolated from Escherichia coli (E. coli) 
cells as disclosed in U.S. Pat. No. 5,212,091. This form of TFPI has been 
shown to be active in the inhibition of bovine factor Xa and in the 
inhibition of human tissue factor-induced coagulation in plasma. In some 
assays, the E. coil-produced TFPI has been shown to be more active than 
TFPI derived from SK hepatoma cells. Methods have been disclosed for 
purification of recombinant TFPI from yeast cell culture medium, such as 
in Petersen et al, J. Biol. Chem. 18:13344-13351 (1993). Truncated forms 
of recombinant TFPI have also been studied, as described in Hamamoto et 
al. J. Biol. Chem. 268:8704-8710 (1993) and Petersen et al. ibid. 
Petersen et al. ibid have attempted to produce TFPI and variants of TFPI, 
including: 1) variants of TFPI in which the complete C-terminal one third 
of the polypeptide, including Kunitz-type domain 3, was deleted; 2) 
variants of TFPI in which just Kunitz-type domain 3 was deleted; and 3) 
variants of TFPI in which the basic portion of the peptide, C-terminal to 
Kunitz-type domain 3, were deleted. They found that high yields were 
obtained only with the first variant. This variant was heterogeneously 
glycosylated, and its anti-coagulant activity was 5-50 fold lower than 
full-length TFPI obtained from mammalian cells. 
A need exists, therefore, for a method to produce in high yield, a protein 
molecule that possesses at least the equivalent, if not enhanced, 
anticoagulant and other activities, of TFPI and that has reduced 
glycosylated moieties that would in turn result in reduced immunogenicity 
of the protein upon administration to a mammal. 
SUMMARY OF THE INVENTION 
It is, therefore, one of the objects of the present invention to provide a 
protein that possesses equivalent or enhanced anticoagulant activity, in 
particular, factor VIIa/TF complex and/or factor Xa inhibitory activity, 
as compared to full length TFPI obtained from mammalian cells, yeast cells 
or bacterial cells. 
Another object of the present invention is to produce in high yield, a 
protein having factor VIIa/TF complex and/or factor Xa inhibitory 
activity. 
Another object of the present invention is to provide a protein having the 
activity of TFPI, TFPI-2, or both whichs target cell surfaces as well or 
better than TFPI or TFPI-2. 
It is further one of the objects of the present invention to provide a 
protein that has reduced glycosylated moieties as compared to the variants 
or full length TFPI described in Petersen et al, ibid.

DETAILED DESCRIPTION OF THE INVENTION 
As used herein, the term "TFPI" refers to the coagulation inhibitor Tissue 
Factor Pathway Inhibitor, also known as Lipoprotein Associated Coagulation 
Inhibitor (LACI), Tissue Factor Inhibitor (TFI) and Extrinsic Pathway 
Inhibitor (EPI). The nucleotide sequence encoding TFPI and the predicted 
amino acid sequence of TFPI have been disclosed in U.S. Pat. No. 
4,966,852, which is herein incorporated by reference. 
As used herein, the term "TFPI-2" refers to a coagulation inhibitor, the 
nucleotide sequence and predicted amino acid sequence of which have been 
reported by Sprecher et al, Proc. Nat. Acad. Sci. U.S.A. (1994) 
91:3353-3357. The disclosure of Sprecher et al is herein incorporated by 
reference. 
As used herein, the term "factor VIIa/TF/Xa binding protein" refers to 
proteins capable of binding to the factor VIIa/TF complex thereby 
inhibiting the function of the complex and further capable of binding 
factor Xa thereby inhibiting its function. The factor VIIa/TF/Xa binding 
proteins contain one or more Kunitz-type domains derived from TFPI (or 
muteins thereof) and one or more Kunitz-type domains from derived TFPI-2 
(or muteins thereof). 
As used herein, the term "first Kunitz-type domain" refers to amino acids 
______________________________________ 
Cys Ala Phe Lys Ala Asp Asp Gly Pro Cys Lys Ala Ile Met 
Lys Arg Phe Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu 
Phe Ile Tyr Gly Gly Cys Glu Gly Asn Gln Asn Arg Phe Glu 
Ser Leu Glu Glu Cys Lys Lys Met Cys [SEQ ID NO: 1] 
______________________________________ 
of TFPI and the amino acid sequence 
______________________________________ 
Cys Leu Leu Pro Leu Asp Tyr Gly Pro Cys Arg Ala Leu Leu 
Leu Arg Tyr Tyr Tyr Asp Arg Tyr Thr Gln Ser Cys Arg Gln 
Phe Leu Tyr Gly Gly Cys Glu Gly Asn Ala Asn Asn Phe Tyr 
Thr Trp Glu Ala Cys Asp Asp Ala Cys [SEQ ID NO: 2] 
______________________________________ 
of TFPI-2; the term "second Kunitz-type domain" refers to amino acids 
______________________________________ 
Cys Phe Leu Glu Glu Asp Pro Gly Ile Cys Arg Gly Tyr Ile 
Thr Arg Tyr Phe Tyr Asn Asn Gln Thr Lys Gln Cys Glu Arg 
Phe Lys Tyr Gly Gly Cys Leu Gly Asn Met Asn Asn Phe Glu 
Thr Leu Glu Glu Cys Lys Asn Ile Cys [SEQ ID NO: 3] 
______________________________________ 
of TFPI and amino acids 
______________________________________ 
Cys Arg Leu Gln Val Ser Val Asp Asp Gln Cys Glu Gly Ser 
Thr Glu Lys Tyr Phe Phe Asn Leu Ser Ser Met Thr Cys Glu 
Lys Phe Phe Ser Gly Gly Cys His Arg Asn Arg Ile Glu Asn 
Arg Phe Pro Asp Glu Ala Thr Cys Met Gly Phe Cys 
[SEQ ID NO: 4] 
______________________________________ 
of TFPI-2; and the term "third Kunitz-type domain" refers to amino acids 
______________________________________ 
Cys Leu Thr Pro Ala Asp Arg Gly Leu Cys Arg Ala Asn Glu 
Asn Arg Phe Tyr Tyr Asn Ser Val Ile Gly Lys Cys Arg Pro 
Phe Lys Tyr Ser Gly Cys Gly Gly Asn Glu Asn Asn Phe Thr 
Ser Lys Gln Glu Cys Leu Arg Ala Cys [SEQ ID NO: 5] 
______________________________________ 
of TFPI and amino acids 
______________________________________ 
Cys Tyr Ser Pro Lys Asp Glu Gly Leu Cys Ser Ala Asn Val 
Thr Arg Tyr Tyr Phe Asn Pro Arg Tyr Arg Thr Cys Asp Ala 
Phe Thr Tyr Thr Gly Cys Gly Gly Asn Asp Asn Asn Phe Val 
Ser Arg Glu Asp Cys Lys Arg Ala Cys [SEQ ID NO: 6] 
______________________________________ 
of TFPI-2. 
As used herein, the term "C-terminal tail" refers to the amino acid 
sequences which are carboxy-terminal to the third Kunitz-type domain of 
TFPI or of TFPI-2, i.e., 
______________________________________ 
Lys Lys Gly Phe Ile Gln Arg Ile Ser Lys Gly Gly Leu Ile 
Lys Thr Lys Arg Lys Arg Lys Lys Gln Arg Val Lys Ile Ala 
Tyr Glu Glu Ile Phe Val Lys Asn Met [SEQ ID NO: 7] 
______________________________________ 
for TFPI and 
______________________________________ 
Ala Lys Ala Leu Lys Lys Lys Lys Lys Met Pro Lys Leu Arg 
Phe Ala Ser Arg Ile Arg Lys Ile Arg Lys Lys Gln Phe 
[SEQ ID NO: 8] 
______________________________________ 
for TFPI-2. These sequences are highly basic and may be involved in cell 
surface localization by glycosaminoglycan (including heparin) or 
phospholipid binding. Further description and explanation of Kunitz-type 
domains 1, 2 and 3 and the P.sub.1 -reactive site for each domain may be 
found in Girard et al, Nature, 338:518-520 (1989). 
As used herein, the term "P.sub.1 -reactive site" refers to the active site 
cleft of a Kunitz-type domain. Alteration of the amino acid residue 
present in the P.sub.1 position can profoundly alter the binding, and 
therefore the inhibitory effect, of the Kunitz-type domain to its target 
protease. 
As used herein, the term "chimeric protein" refers to a polypeptide 
consisting of one or more domains from different proteins or mutations 
within a single protein giving the characteristics of another protein. For 
example, a chimeric protein as used herein would include a factor 
VIIa/TF/Xa binding protein containing SEQ ID NO: 2 and SEQ ID NO: 1. 
As used herein, the term "mutein" refers to a normal or wild-type sequence 
in which 1-5 amino acid substitutions have been made. For example, a 
mutein in Kunitz-type domain 1 of TFPI [SEQ ID NO: 1] may be made in the 
P.sub.1 position by changing a lysine residue to an arginine residue. This 
substitution has the effect of altering the properties of Kunitz-type 
domain 1 of TFPI, including affinity for factor VIIa/TF complex, to those 
of Kunitz-type domain 1 of TFPI-2. 
As used herein, the term "pharmaceutically acceptable carrier" refers to a 
medium which does not interfere with the effectiveness of the biological 
activity of the active ingredient and which is not toxic to the hosts to 
which it is administered. 
As used herein, the term "pharmacologically effective amount" refers to the 
amount of protein administered to the host that results in reduction of 
morbidity and mortality resulting from the condition being treated. 
Conditions that may be treated include sepsis, septic shock and thrombosis 
disorders, including thrombosis during and after microsurgery and 
thrombosis from abrupt reclosure after angioplasty. The exact amount 
administered depends on condition, severity, subject etc., but may be 
determined by routine methods. The term "pharmacologically effective 
amount" also refers to the amount of protein administered to the host that 
prevents morbidity and mortality resulting associated with a condition. 
Conditions that may be prevented include sepsis, septic shock and 
thrombosis disorders, including thrombosis during and after microsurgery 
and thrombosis from abrupt reclosure after angioplasty. The exact amount 
administered depends on condition, severity, subject etc., but may be 
determined by routine methods. 
Factor VIIa/TF/Xa binding proteins of the invention include muteins of TFPI 
and TFPI-2, the muteins having single or multiple amino acid 
substitutions. Muteins within the scope of this definition include: (a) 
TFPI or TFPI-2 muteins having 1-5 conservative amino acid substitutions 
that do not substantially change the conformation of the molecule; (b) 
TFPI or TFPI-2 muteins with amino acid substitutions that eliminate one or 
more of the three sites for N-linked glycosylation; (c) TFPI muteins 
having 1-5 amino acid substitutions that change a residue of TFPI to a 
corresponding residue of TFPI-2; (d) TFPI-2 muteins having 1-5 amino acid 
substitutions that change a residue of TFPI-2 to a corresponding residue 
of TFPI; (e) TFPI or TFPI-2 muteins with amino acid substitutions in 
P.sub.1 reactive sites in one or more Kunitz-type domains; and (f) TFPI or 
TFPI-2 muteins with amino acid substitutions at positions within 5 amino 
acids of the P.sub.1 reactive sites in one or more Kunitz-type domains. In 
a preferred embodiment, the lysine residue in the P.sub.1 -reactive site 
of the first Kunitz-type domain of TFPI [SEQ ID NO: 1] is replaced with 
arginine. The mutein has the following sequence: 
______________________________________ 
Asp Ser Glu Glu Asp Glu Glu His Thr Ile Ile Thr Asp Thr 
Glu Leu Pro Pro Leu Lys Leu Met His Ser Phe Cys Ala Phe 
Lys Ala Asp Asp Gly Pro Cys Arg Ala Ile Met Lys Arg Phe 
Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu Phe Ile Tyr 
Gly Gly Cys Glu Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu 
Glu Cys Lys Lys Met Cys Thr Arg Asp Asn Ala Asn Arg Ile 
Ile Lys Thr Thr Leu Gln Gln Glu Lys Pro Asp Phe Cys Phe 
Leu Glu Glu Asp Pro Gly Ile Cys Arg Gly Tyr Ile Thr Arg 
Tyr Phe Tyr Asn Asn Gln Thr Lys Gln Cys Glu Arg Phe Lys 
Tyr Gly Gly Cys Leu Gly Asn Met Asn Asn Phe Glu Thr Leu 
Glu Glu Cys Lys Asn Ile Cys Glu Asp Gly Pro Asn Gly Phe 
Gln Val Asp Asn Tyr Gly Thr Gln Leu Asn Ala Val Asn Asn 
Ser Leu Thr Pro Gln Ser Thr Lys Val Pro Ser Leu Phe Glu 
Phe His Gly Pro Ser Trp Cys Leu Thr Pro Ala Asp Arg Gly 
Leu Cys Arg Ala Asn Glu Asn Arg Phe Tyr Tyr Asn Ser Val 
Ile Gly Lys Cys Arg Pro Phe Lys Tyr Ser Gly Cys Gly Gly 
Asn Glu Asn Asn Phe Thr Ser Lys Gln Glu Cys Leu Arg Ala 
Cys Lys Lys Gly Phe Ile Gln Arg Ile Ser Lys Gly Gly Leu 
Ile Lys Thr Lys Arg Lys Arg Lys Lys Gln Arg Val Lys Ile 
Ala Tyr Glu Glu Ile Phe Val Lys Asn Met. [SEQ ID NO: 9]. 
______________________________________ 
Muteins of TFPI and TFPI-2 containing one or more amino acid substitutions 
may be prepared by appropriate mutagenesis of the sequence of a 
recombinant cloning vehicle encoding TFPI or TFPI-2, using techniques 
known to those skilled in the art. Techniques for mutagenesis include, 
without limitation, site specific mutagenesis. Site specific mutagenesis 
can be carried out using any number of procedures known in the art. These 
techniques are described by Smith, Annual Review of Genetics, 19:423 
(1985), and modifications of some of the techniques are described in 
Methods in Enzymology, 154, part E, (eds.) Wu and Grossman (1987), 
chapters 17, 18, 19, and 20. A preferred procedure when using site 
specific mutagenesis is a modification of the Gapped Duplex site directed 
mutagenesis method. The general procedure is described by Kramer, et al., 
in chapter 17 of the Methods in Enzymology, above. Another technique for 
generating point mutations in a nucleic acid sequence is the use of PCR 
techniques, including overlapping PCR, as described in PCR PROTOCOLS: A 
GUIDE TO METHODS AND APPLICATIONS, (eds.) Innis, Gelfand, Sninsky and 
White (Academic Press, 1990). 
The muteins of TFPI and TFPI-2 may also be truncated at the end of the 
second Kunitz-type domain. Such truncated molecules retain the ability to 
bind factor VIIa/TF complex and Xa yet can be expressed at higher levels 
in such organisms as yeast. The truncated TFPI and TFPI-2 muteins will 
likely lead to enhanced recovery of a product containing correctly folded 
Kunitz-type domains due to the removal of six cysteine residues in the 
third Kunitz-type domain. The truncated muteins may also have the tail 
sequence of TFPI or of TFPI-2 attached at the carboxy-terminal end to give 
the mutein cell surface-binding ability, preferably by binding to 
glycosaminoglycans (including heparin) or phospholipid at the cell 
surface. 
Chimeric proteins capable of binding factor VIIa/TF and factor X.sub.a and 
containing various portions of TFPI or TFPI-2 are also within the scope of 
the invention. One class of proteins within the scope of the invention can 
be represented by the following generic formula: 
EQU A--(X.sub.1).sub.a --B--(X.sub.2).sub.b --C 
wherein A and C are optional flanking peptides, the flanking peptides 
independently containing 1-100 amino acids; 
wherein B is an optional spacer peptide, the spacer peptide containing 1-25 
amino acids; 
wherein each X.sub.1 is --D--K.sub.1 --E-- 
where D,E are peptides of 1-25 amino acids, 
where K.sub.1 is independently Kunitz-type domain 1 from TFPI or TFPI-2 
[SEQ ID NO: 1 or SEQ ID NO: 2] or a mutein of the aforementioned 
Kunitz-type domains; 
wherein each X.sub.2 is --F--K.sub.2 --G-- 
where F,G are peptides of 1-25 amino acids, 
where K.sub.2 is independently Kunitz-type domain 2 from TFPI or TFPI-2 
[SEQ ID NO: 3 or SEQ ID NO: 4] or a mutein of the aforementioned 
Kunitz-type domains; 
wherein a,b are integers from 0-6; and 
wherein the molecule is not native TFPI or TFPI-2. 
A,B,C,D,E,F,G may independently comprise portions of native TFPI or TFPI-2 
sequences. For example, B, D, E, F, and G may independently comprise 
peptide sequences between Kunitz-type domains 1 and 2 of TFPI or TFPI-2, 
or peptide sequences between Kunitz-type domains 2 and 3 of TFPI and 
TFPI-2. The flanking peptides A and C may also have cell surface 
localization properties and may be the C-terminal tail sequence from TFPI 
or TFPI-2 [SEQ ID NO: 7 or SEQ ID NO: 8]. Alternatively, other cell 
surface localizing peptide sequences may be used. These sequences 
preferably have glycosaminoglycan binding ability and, most preferably, 
bind heparin. Such peptide sequences may be derived from proteins having 
heparin binding activity including, but not limited to, the following: 
protease nexin-1, protease nexin-2, antithrombin III, protein C inhibitor, 
platelet factor 4, heparin cofactor II, ghilanten-related inhibitors, and 
bovine pancreatic trypsin inhibitor. Appropriate portions of these 
proteins (i.e. those with glycosaminoglycan binding activity) may be 
attached in the A or C position (or both). 
In the case of TFPI, the appropriate portion may be the C-terminal tail 
[SEQ ID NO: 7] or 
______________________________________ 
Lys Thr Lys Arg Lys Arg Lys Lys Gln Arg Val Lys Ile Ala 
Tyr Glu Glu Ile Phe Val Lys Asn Met. [SEQ ID NO: 10]. 
______________________________________ 
In the case of TFPI-2, the appropriate portion may be the C-terminal tail 
[SEQ ID NO: 8] or 
______________________________________ 
Lys Lys Lys Lys Lys Met Pro Lys Leu Arg Phe Ala Ser Arg 
Ile Arg Lys Ile Arg Lys Lys Gln Phe. [SEQ ID NO: 11]. 
______________________________________ 
In the case of antithrombin III, the appropriate portion may be 
______________________________________ 
Ala Lys Leu Asn Cys Arg Leu Tyr Arg Lys Ala Asn Lys Ser 
Ser Lys Leu. [SEQ ID NO: 12]. 
______________________________________ 
The appropriate portion of antithrombin III may also be 
______________________________________ 
Thr Ser Asp Gln Ile His Phe Phe Phe Ala Lys Leu Asn Cys Arg. 
[SEQ ID NO: 13] 
______________________________________ 
In the case of protein C inhibitor, the appropriate portion may be: 
______________________________________ 
Ser Glu Lys Thr Leu Arg Lys Trp Leu Lys Met Phe Lys Lys 
Arg Glu Leu Glu Glu Tyr. [SEQ ID NO: 14]. 
______________________________________ 
The appropriate portion of protein C inhibitor may be: 
______________________________________ 
His Arg His His Pro Arg Glu Met Lys Lys Arg Val Glu Asp 
Leu. [SEQ ID NO: 15]. 
______________________________________ 
In the case of heparin cofactor II, the appropriate portion may be 
______________________________________ 
Phe Arg Lys Leu Thr His Arg Leu Phe Arg Arg Asn Phe Gly 
Tyr Thr Leu Arg. [SEQ ID NO: 16]. 
______________________________________ 
In the case of platelet factor 4, the appropriate portion may be 
______________________________________ 
Leu Tyr Lys Lys Ile Leu Lys Lys Leu Leu Glu Ala. 
[SEQ ID NO: 17]. 
______________________________________ 
In the case of ghilanten-related inhibitors, the appropriate portion may be 
______________________________________ 
Asn Gly Leu Lys Arg Asp Lys Leu Gly Cys Glu Tyr Cys Glu 
Cys Arg Pro Lys Arg Lys Leu Ile Pro Arg Leu Ser. 
[SEQ ID NO: 18]. 
______________________________________ 
In a preferred embodiment, a factor VIIa/TF/X.sub.a binding protein 
contains the first Kunitz-type domain [SEQ ID NO: 2], including the 
amino-terminal sequence, of TFPI-2, the second Kunitz-type domain of TFPI 
[SEQ ID NO: 3], the third Kunitz-type domain of TFPI [SEQ ID NO: 5] and/or 
the TFPI C terminal tail sequence[SEQ ID NO: 7]. One skilled in the art 
will appreciate that this molecule is but one of numerous species that may 
be produced and that various portions of the peptide sequences linking the 
Kunitz-type domains from TFPI or from TFPI-2 may be included in the 
molecule. 
Also within the scope of the invention are factor VIIa/TF/X.sub.a binding 
proteins containing two or more of the same Kunitz-type domain from TFPI 
or from TFPI-2. For example, factor VIIa/TF/X.sub.a binding proteins 
comprising two or more iterations of the first Kunitz-type domain of 
TFPI-2 [SEQ ID NO: 2] may prepared. Such molecules may be particularly 
useful increased inhibition of factor VIIa/TF complex. Factor 
VIIa/TF/X.sub.a binding proteins containing two or more iterations of the 
second Kunitz-type domain of TFPI [SEQ ID NO: 3] may also be prepared. 
Such molecules may be particularly useful for the increased inhibition of 
factor X.sub.a by the protein. A preferred factor VIIa/TF/Xa binding 
protein containing more than one iteration of the same Kunitz-type domain 
is represented by the formula: 
EQU A--[X.sub.1 --B--X.sub.2 ].sub.c --C 
wherein A and C are optional flanking peptides, the flanking peptides 
independently containing 1-100 amino acids; 
wherein B is an optional spacer peptide, the spacer peptide containing 1-25 
amino acids; 
wherein each X.sub.1 is --D--K.sub.1 --E-- 
where D,E arc peptides of 1-25 amino acids, 
where K.sub.1 is Kunitz-type domain 1 from TFPI or TFPI-2 [SEQ ID NO: 1 or 
SEQ ID NO: 2] or a mutein of the aforementioned Kunitz-type domains; 
wherein each X.sub.2 is --F--K.sub.2 --G-- 
where F,G are peptides of 1-25 amino acids, 
where K.sub.2 is Kunitz-type domain 2 from TFPI or TFPI-2 [SEQ ID NO: 3 or 
SEQ ID NO: 4] or a mutein of the aforementioned Kunitz-type domains; 
wherein c is an integer from 1-10. 
A, B, C, D, E, F, and G may also have the same sequences disclosed above in 
reference to the generic structure. The factor VIIa/TF/Xa binding protein 
may be one in which K.sub.1 and K.sub.2 are SEQ ID NO: 2 and SEQ ID NO: 3. 
One skilled in the art of DNA cloning and in possession of the DNA encoding 
TFPI and TFPI-2 is able to prepare suitable DNA molecules for production 
of such chimeric proteins using known cloning procedures (e.g. restriction 
enzyme digestion of TFPI and TFPI-2 encoding DNA, exonuclcase digestion, 
ligation, and other appropriate procedures outlined in any of the 
following: Sambrook, et al., MOLECULAR CLONING: A LABORATORY MANUAL 2nd 
ed. (Cold Spring Harbor Laboratory Press, 1989); DNA CLONING, Vol. I and 
II, D. N. Glover ed. (IRL Press, 1985); OLIGONUCLEOTIDE SYNTHESIS, M. J. 
Gait ed. (IRL Press, 1984); NUCLEIC ACID HYBRIDIZATION, B. D. Hames & S. 
J. Higgins eds. (IRL Press, 1984); TRANSCRIPTION AND TRANSLATION, B. D. 
Hames & S. J. Higgins eds., (IRL Press, 1984); ANIMAL CELL CULTURE, R. I. 
Freshney ed. (IRL Press, 1986); IMMOBILIZED CELLS AND ENZYMES, K. Mosbach 
(IRL Press, 1986); B. Perbal, A PRACTICAL GUIDE TO MOLECULAR CLONING, 
Wiley (1984); the series, METHODS IN ENZYMOLOGY, Academic Press, Inc.; 
GENE TRANSFER VECTORS FOR MAMMALIAN CELLS, J. H. Miller and M. P. Calos 
eds. (Cold Spring Harbor Laboratory, 1987); METHODS IN ENZYMOLOGY, Vol. 
154 and 155, Wu and Grossman, eds., and Wu, ed., respectively (Academic 
Press, 1987), IMMUNOCHEMICAL METHODS IN CELL AND MOLECULAR BIOLOGY, R. J. 
Mayer and J. H. Walker, eds. (Academic Press London, Harcourt Brace U.S., 
1987), PROTEIN PURIFICATION: PRINCIPLES AND PRACTICE, 2nd ed. 
(Springer-Verlag, N.Y. (1987), and HANDBOOK OF EXPERIMENTAL IMMUNOLOGY, 
Vol. I-IV, D. M. Weir et al., (Blackwell Scientific Publications, 1986); 
Kitts et al., Biotechniques 14:810-817 (1993); Munemitsu et al., Mol. 
Cell. Biol. 10:5977-5982 (1990). Alternatively, the entire sequence or 
portions of nucleic acid sequences encoding proteins described above may 
be prepared by synthetic methods (e.g. using DNA synthesis machines). 
Finally, a preferred method of preparing nucleic acid molecules encoding 
the described chimeric proteins is by use of PCR techniques as described 
in Innis et al, supra. 
The proteins described above may be prepared using any suitable expression 
system including, without limitation, the following expression systems: 
mammalian tissue culture, insect cell culture, bacterial cell culture and 
yeast cell culture. Mammalian expression systems are known in the art. 
Sambrook et al. (1989) "Expression of Cloned Genes in Mammalian Cells." In 
Molecular Cloning: A Laboratory Manual, 2nd ed. Mammalian cell lines 
available as hosts for expression are known in the art and include many 
immortalized cell lines available from the American Type Culture 
Collection (ATCC). TFPI has been isolated from human plasma and from human 
tissue culture cells including HepG2, Chang liver and SK hepatoma cells. 
Recombinant TFPI has been expressed in mouse C127 cells, baby hamster 
kidney cells, Chinese hamster ovary cells and human SK hepatoma cells. 
The proteins of the invention may also be produced in insect cells using a 
vector containing baculovirus sequences. Materials and methods for 
baculovirus/insect cell expression systems are commercially available in 
kit form from, inter alia, Invitrogen, San Diego Calif. ("MaxBac" kit). 
These techniques are generally known to those skilled in the art and fully 
described in Summers and Smith, Texas Agricultural Experiment Station 
Bulletin No. 1555 (1987) hereinafter "Summers and Smith"). Currently, the 
most commonly used transfer vector for introducing foreign genes into 
AcNPV is pAc373. Many other vectors, known to those of skill in the art, 
have also been designed. These include, for example, pVL985 (which alters 
the polyhedrin start codon from ATG to ATT, and which introduces a BamHI 
cloning site 32 base pairs downstream from the ATT; see Luckow and 
Summers, Virology (1989) 17:31. Methods tier introducing heterologous DNA 
into the desired site in the baculovirus virus are known in the art. (See 
Summers and Smith supra; Ju et al. (1987); Smith et al, Mol. Cell. Biol. 
(1983) 3:2156; and Luckow and Summers (1989)). For example, the insertion 
can be into a gene such as the polyhedrin gene, by homologous double 
crossover recombination; insertion can also be into a restriction enzyme 
site engineered into the desired baculovirus gene. Miller et al., 
Bioessays 4:91 (1989). The DNA sequence, when cloned in place of the 
polyhedrin gene in the expression vector, is flanked both 5' and 3' by 
polyhedrin-specific sequences and is positioned downstream of the 
polyhedrin promoter. 
Recombinant baculovirus expression vectors have been developed for 
infection into several insect cells. For example, recombinant 
baculoviruses have been developed for, inter alia: Aedea aegypti, Bombyx 
mori, Drosophila melanogaster, Spodoptera frugiperda, and Trichoplusia ni 
(PCT Pub. No. WO 89/046699; Carbonell et al., J. Virol. 56:153 (1985); 
Wright Nature 321:718 (1986); Smith et al., Mol. Cell. Biol. 3:2156 
(1983); and see generally, Fraser, et al. Cell. Dev. Biol. 25:225 (1989). 
Cells and cell culture media are commercially available for both direct 
and fusion expression of heterologous polypeptides in a 
baculovirus/expression system; cell culture technology is generally known 
to those skilled in the art. See, e.g., Summers and Smith supra. The 
modified insect cells may then be grown in an appropriate nutrient medium, 
which allows for stable maintenance of the plasmid(s) present in the 
modified insect host. A presently preferred media is described in EPO 380 
495. 
Numerous bacterial expression techniques are known in the art. Sambrook et 
al. (1989) "Expression of cloned genes in Escherichia coli." In Molecular 
Cloning: A Laboratory Manual. Expression and transformation vectors, 
either extra-chromosomal replicons or integrating vectors, have been 
developed for transformation into many bacteria. For example, expression 
vectors have been developed for, inter alia, the following bacteria: 
Bacillus subtilis (Palva et al. (1982) Proc. Natl. Acad. Sci. U.S.A. 
79:5582; EPO 036 259 and 063 953; PCT WO 84/04541), E. coli (Shimatake et 
al. (1981) Nature 292:128; Amann et al. (1985) Gene 40:183; Studier et al. 
(1986) J. Mol. Biol. 189:113; EPO Publ. Nos. 036 776, 136 829 and 136 
907), Streptococcus cremoris (Powell et al. (1988) Appl. Environ. 
Microbiol. 54:655); Streptococcus lividans (Powell et al. (1988) Appl. 
Environ. Microbiol. 54:655), Streptomyces lividans (U.S. Pat. No. 
1,745,056). 
The DNA encoding the protein of the present invention may be joined to a 
signal peptide liar export or secretion of the mature protein to the 
periplasmic space of bacteria, using techniques that are conventional in 
the art. Moreover, transcription and translation can further be optimized 
in a bacterial expression system by varying the spacing between the DNA to 
be expressed and the sequences encoding the promoter and ribosome binding 
site. 
Yeast expression systems are also known in the art. Fusion proteins provide 
one means for expression of the proteins of the invention in yeast 
systems. Usually, a DNA sequence encoding the N-terminal portion of an 
endogenous yeast protein, or other stable protein, is fused to the 5' end 
of a heterologous coding sequence. Upon expression, this construct will 
provide a fusion of the two amino acid sequences. The DNA sequence at the 
junction of the two amino acid sequences may or may not encode a cleavable 
site. See e.g., EPO 196 056. A preferred fusion protein is a ubiquitin 
fusion protein. Such a fusion protein is made with the ubiquitin region 
that preferably retains a site for a processing enzyme, which allows a 
ubiquitin-specific processing protease to cleave the ubiquitin from the 
foreign protein. Through this method, therefore, foreign protein with an 
authentic amino terminus can be isolated from within the yeast cell. 
Production of ubiquitin fusion proteins is described in co-pending U.S. 
patent application Ser. Nos. 07/806,813 and 07/957,627. This method is 
reviewed in Bart et al, in RECOMBINANT SYSTEMS IN PROTEIN EXPRESSION 
(Elsevier Science Publishers B.V., 1991), pp. 37-46. 
Alternatively, foreign proteins can be secreted from the cell into the 
growth media by creating chimeric DNA molecules that encode a fusion 
protein comprised of a leader sequence fragment that provide for secretion 
in yeast of the foreign protein. Preferably, there are processing sites 
encoded between the leader fragment and the foreign gene that can be 
cleaved either in vivo or in vitro. The leader sequence fragment usually 
encodes a signal peptide comprised of hydrophobic amino acids which direct 
the secretion of the protein from the cell. 
DNA encoding suitable signal sequences can be derived from genes for 
secreted yeast proteins, such as the yeast invertase gene (EP 012 873; JPO 
62,096,086), the .alpha.-factor gene (U.S. Pat. Nos. 4,588,684 and 
4,870,008; EP 116,201) and truncated versions of the .alpha.-factor gene 
as described in EP 324 274 and co-pending U.S. patent application Ser. No. 
07/864,206. Alternatively, leaders of non-yeast origin, such as an 
interferon leader, exist that also provide for secretion in yeast (EP 060 
057). Preferably, the .alpha.-factor gene is used in nucleic acid 
constructs designed for secretion of the proteins of the invention. 
Another useful class of secretion leaders are those that employ a fragment 
of the yeast .alpha.-factor gene, which contains both a "pre" signal 
sequence, and a "pro" region. The types of .alpha.-factor fragments that 
can be employed include the full-length pre-pro .alpha. factor leader 
(about 83 amino acid residues) as well as truncated .alpha.-factor leaders 
(usually about 25 to about 50 amino acid residues) (U.S. Pat. Nos. 
4,546,083 and 4,870,008; EP 324 274). Additional leaders employing an 
.alpha.-factor leader fragment that provides for secretion include hybrid 
.alpha.-factor leaders made with a presequence of a first yeast, but a 
pro-region from a second yeast .alpha.-factor. (See e.g., PCT WO 
89/02463.) 
Expression vectors encoding the proteins of the invention are often 
maintained in a replicon, such as an extrachromosomal element (e.g., 
plasmid) capable of stable maintenance in a host, such as yeast or 
bacteria. The replicon may have two replication systems, thus allowing it 
to be maintained, for example, in yeast for expression and in a 
procaryotic host for cloning and amplification. Examples of such 
yeast-bacteria shuttle vectors include YEp24 (Botstein et al, Gene 8:17-24 
(1979)), pC1/1 (Brake et al, Proc. Natl. Acad. Sci U.S.A. 81:4642-4646 
(1984)), and YRp17 (Stinchcomb et al, J. Mol. Biol. 158:157 (1982)). In 
addition, a replicon may be either a high or low copy number plasmid. A 
high copy number plasmid will generally have a copy number ranging from 
about 5 to about 200, and usually about 10 to about 150. A host containing 
a high copy number plasmid will preferably have at least about 10, and 
more preferably at least about 20. Either a high or low copy number vector 
may be selected, depending upon the effect of the vector and the foreign 
protein on the host. See e.g., Brake et al, supra. For production of the 
proteins of the invention in a yeast cell wherein the protein is retained 
within the yeast cell, a plasmid such as pAB24 may be used. Sabin et al, 
(1989) Bio/Technology 2:705-709. pAB24 contains a GAP/ADH hybrid promoter, 
containing portions of an ADH promoter capable of directing high levels of 
expression of the sequences under its control but which also contains GAP 
regulatory sequences, allowing expression of the same sequence a desired 
point in the growth of a culture. 
Alternatively, the expression constructs can be integrated into the host 
genome with an integrating vector. Integrating vectors usually contain at 
least one sequence homologous to a host chromosome that allows the vector 
to integrate, and preferably contain two homologous sequences flanking the 
expression construct. Integrations appear to result from recombinations 
between homologous DNA in the vector and the host chromosome. Orr-Weaver 
et al, Meth. Ezymol. 101:228-245 (1983). An integrating vector may be 
directed to a specific locus in yeast by selecting the appropriate 
homologous sequence for inclusion in the vector. See Orr-Weaver et al, 
supra. One or more expression construct may integrate, possibly affecting 
levels of recombinant protein produced. Rine et al, Proc. Natl. Acad. Sci. 
U.S.A. 80:6750 (1983). The chromosomal sequences included in the vector 
can occur either as a single segment in the vector, which results in the 
integration of the entire vector, or two segments homologous to adjacent 
segments in the chromosome and flanking the expression construct in the 
vector, which can result in the stable integration of only the expression 
construct. 
Usually, extrachromosomal and integrating expression constructs may contain 
selectable markers to allow for the selection of yeast strains that have 
been transformed. Selectable markers may include biosynthetic genes that 
can be expressed in the yeast host, such as ADE2, HIS4, LEU2, TRP1, and 
ALG7, and the G418 resistance gene, which confer resistance in yeast cells 
to tunicamycin and G418, respectively. In addition, a suitable selectable 
marker may also provide yeast with the ability to grow in the presence of 
toxic compounds, such as metal. For example, the presence of CUP1 allows 
yeast to grow in the presence of copper ions (Butt et al, Microbiol. Rev. 
51:351 (1987)). 
Alternatively, some of the above described components can be put together 
into transformation vectors. Transformation vectors are usually comprised 
of a selectable marker that is either maintained in a replicon or 
developed into an integrating vector, as described above. 
Expression and transformation vectors, either extrachromosomal replicons or 
integrating vectors, have been developed for transformation into many 
yeasts. For example, expression vectors have been developed for, inter 
alia, the following yeasts: Candida albicans (Kurtz, et al, Mol. Cell. 
Biol. 6:142 (1986)), Candida maltosa (Kunze, et al, J. Basic Microbiol. 
25:141 (1985)), Hansenula polymorpha (Gleeson, et al, J. Gen. Microbiol. 
132:3459 (1986); Roggenkamp et al, Mol. Gen. Genet. 202:302 (1986)), 
Kluyveromyces fragilis (Das, et al, J. Bacteriol. 158:1165 (1984)), 
Kluyveromyces lactis (De Louvencourt et al, J. Bacteriol. 154:737 (1983); 
Van den Berg et al, Bio/Technology 8:135 (1990)), Pichia guillerimondii 
(Kunze et al, J. Basic Microbiol. 25:141 (1985)), Pichia pastoris (Cregg, 
et al, Mol. Cell. Biol. 5:3376 (1985); U.S. Pat. Nos. 4,837,148 and 
4,929,555), Saccharomyces cerevisiae (Hinnen et al, Proc. Natl. Acad. Sci. 
U.S.A. 75:1929 (1978); Ito et al, J. Bacteriol. 153:163 (1983)), 
Schizosaccharomyces pombe (Beach and Nurse, Nature 300:706 (1981)), and 
Yarrowia lipolytica (Davidow, et al, Curr. Genet. 10:380471 (1985) and 
Gaillardin et al, Curr. Genet. 10:49 (1985)). 
Transformation procedures that may be used herein to transform yeast cells 
include electropotation, as described in "Guide to Yeast Genetics and 
Molecular Biology," Vol 194 METHODS IN ENZYMOLOGY, C. Guthrie and G. R. 
Fink, (Academic Press 1991). Other procedures include the transformation 
of spheroplasts or the transformation of alkali cation-treated intact 
cells. Such procedures are described in, for example, Kurtz et al, Mol. 
Cell. Biol. 6:142 (1986); Kunze et al, J. Basic Microbiol. 25:141 (1985), 
for Candida; Gleeson et al, J. Gen. Microbiol. 132:3459 (1986); Roggenkamp 
et al., Mol. Gen. Genet. 202:302, for Hansenula (1986); Das et al, J. 
Bacteriol. 158:1165 (1984); De Louvencourt et al, J. Bacteriol. 154:1165 
(1983); Van den Berg et al, Bio/Technology 8:135 (1990) for Kluyveromyces; 
Cregg et al, Mol. Cell. Biol. 5:3376 (1985); Kunze et al, J. Basic 
Microbiol. 25:141 (1985); U.S. Pat. Nos. 4,837,148 and 4,929,555, for 
Pichia; Hinnen et al, Proc. Natl. Acad. Sci. U.S.A. 75:1929 (1978); Ito et 
al, J. Bacteriol. 153:163 (1983), for Saccharomyces; Beach and Nurse, 
Nature 300:706 (1981), for Schizosaccharomyces; Davidow et al, Curr. 
Genet. 10:39 (1985); Gaillardin et al, Curr. Genet. 10:49 (1985), for 
Yarrowia. 
Yeast cell culture, especially Saccharomyces cerevisiae, are preferred for 
production of the proteins of the invention. In a preferred embodiment, 
the chimeric protein having the primary amino acid sequence: 
__________________________________________________________________________ 
Asp 
Ser 
Glu 
Glu 
Asp 
Glu 
Glu 
His 
Thr 
Ile 
Ile 
Thr 
Asp 
Thr 
Glu 
Leu 
Pro 
Pro 
Leu 
Lys 
Leu 
Met 
His 
Ser 
Phe 
Cys 
Ala 
Phe 
Lys 
Ala 
Asp 
Asp 
Gly 
Pro 
Cys 
Arg 
Ala 
Ile 
Met 
Lys 
Arg 
Phe 
Phe 
Phe 
Asn 
Ile 
Phe 
Thr 
Arg 
Gln 
Cys 
Glu 
Glu 
Phe 
Ile 
Tyr 
Gly 
Gly 
Cys 
Glu 
Gly 
Asn 
Gln 
Asn 
Arg 
Phe 
Glu 
Ser 
Leu 
Glu 
Glu 
Cys 
Lys 
Lys 
Met 
Cys 
Thr 
Arg 
Asp 
Asn 
Ala 
Asn 
Arg 
Ile 
Ile 
Lys 
Thr 
Thr 
Leu 
Gln 
Gln 
Glu 
Lys 
Pro 
Asp 
Phe 
Cys 
Phe 
Leu 
Glu 
Glu 
Asp 
Pro 
Gly 
Ile 
Cys 
Arg 
Gly 
Tyr 
Ile 
Thr 
Arg 
Tyr 
Phe 
Tyr 
Asn 
Gln 
Gln 
Thr 
Lys 
Gln 
Cys 
Glu 
Arg 
Phe 
Lys 
Tyr 
Gly 
Gly 
Cys 
Leu 
Gly 
Asn 
Met 
Asn 
Asn 
Phe 
Glu 
Thr 
Leu 
Glu 
Glu 
Cys 
Lys 
Asn 
Ile 
Cys 
Glu 
Asp 
Gly 
Pro 
Asn 
Gly 
Phe 
Gln 
Val 
Asp 
Asn 
Tyr 
Gly 
Thr 
[SEQ ID NO: 19] 
__________________________________________________________________________ 
is produced in a yeast cell as a fusion protein with .alpha.-factor. 
Alternatively, the flanking peptides may contain sequences derived from 
other protease inhibitors, especially protein inhibitors which act at the 
cell surface. The protein inhibitor protease nexin 1 (hereinafter, nexin) 
is but one example. Nexin is known to bind heparin thereby helping to 
localize nexin on the cell surface. Further, nexin binds to and inhibits 
factor Xa. 
The factor VIIa/TF/X.sub.a binding proteins of the invention may be assayed 
for activity by a prothrombin time clotting assay or a factor Xa 
amidolytic assay (Wun et al, J. Biol. Chem. 265:16096 (1990)) as set forth 
in the Examples below. 
Formulation and Administration 
Factor VIIa/TF/X.sub.a binding proteins of the invention may be 
administered at a concentration that is therapeutically effective to treat 
and prevent septic shock. To accomplish this goal, the factor 
VIIa/TF/X.sub.a binding proteins of the invention are preferably 
administered intravenously. Methods to accomplish this administration are 
known to those of ordinary skill in the art. 
Before administration to patients, formulants may be added to the factor 
VIIa/TF/Xa binding proteins of the invention. A liquid formulation may be 
used. For example, these formulants may include oils, polymers, vitamins, 
carbohydrates, amino acids, salts, buffers, albumin, surfactants, or 
bulking agents. Carbohydrates which may be used in the formulation include 
sugar or sugar alcohols such as mono, di, or polysaccharides, or water 
soluble glucans. The saccharides or glucans can include fructose, 
dextrose, lactose, glucose, mannose, sorbose, xylose, maltose, sucrose, 
dextran, pullulan, dextrin, alpha and beta cyclodextrin, soluble starch, 
hydroxethyl starch and carboxymethylcellulose, or mixtures thereof. 
Sucrose is most preferred. Sugar alcohol is defined as a C.sub.4 to 
C.sub.8 hydrocarbon having an --OH group and includes galactitol, 
inositol, mannitol, xylitol, sorbitol, glycerol, and arabitol. Mannitol is 
most preferred. These sugars or sugar alcohols mentioned above may be used 
individually or in combination. There is no fixed limit to amount used as 
long as the sugar or sugar alcohol is soluble in the aqueous preparation. 
Preferably, the sugar or sugar alcohol concentration is between 1.0 w/v % 
and 7.0 w/v %, more preferable between 2.0 and 6.0 w/v %. Preferably amino 
acids include levorotary (L) forms of carnitine, arginine, and betaine; 
however, other amino acids may be added. Preferred polymers include 
polyvinylpyrrolidone (PVP) with an average molecular weight between 2,000 
and 3,000, or polyethylene glycol (PEG) with an average molecular weight 
between 3,000 and 5,000. It is also preferred to use a buffer in the 
composition to minimize pH changes in the solution before lyophilization 
or after reconstitution. Most any physiological buffer may be used, but 
citrate, phosphate, succinate, and glutamate buffers or mixtures thereof 
are preferred. Most preferred is a citrate buffer. Further, the use of 
sulfates should be avoided in preparation of the formulation. Preferably, 
the concentration is from 0.01 to 0.3 molar. Surfactants that can be added 
to the formulation are shown in EP Nos. 270,799 and 268,110. 
Additionally, the factor VIIa/TF/Xa binding proteins of the invention can 
be chemically modified by covalent conjugation to a polymer to increase 
its circulating half-life, for example. Preferred polymers, and methods to 
attach them to peptides, are shown in U.S. Pat. Nos. 4,766,106, 4,179,337, 
4,495,285, and 4,609,546 which are all hereby incorporated by reference in 
their entireties. Preferred polymers are polyoxyethylated polyols and 
polyethyleneglycol (PEG). PEG is soluble in water at room temperature and 
has the general formula: 
EQU R(O--CH.sub.2 --CH.sub.2).sub.n O--R 
where R can be hydrogen, or a protective group such as an alkyl or alkanol 
group. Preferably, the protective group has between 1 and 8 carbons, more 
preferably it is methyl. The symbol n is a positive integer, preferably 
between 1 and 1,000, more preferably between 2 and 500. The PEG has a 
preferred average molecular weight between 1000 and 40,000, more 
preferably between 2000 and 20,000, most preferably between 3,000 and 
12,000. Preferably, PEG has at least one hydroxy group, more preferably it 
is a terminal hydroxy group. It is this hydroxy group which is preferably 
activated to react with a free amino group on the inhibitor. However, it 
will be understood that the type and amount of the reactive groups may be 
varied to achieve a covalently conjugated PEG/factor VIIa/TF/Xa of the 
present invention. 
Water soluble polyoxyethylated polyols are also useful in the present 
invention. They include polyoxyethylated sorbitol, polyoxyethylated 
glucose, polyoxyethylated glycerol (POG), etc. POG is preferred. One 
reason is because the glycerol backbone of polyoxyethylated glycerol is 
the same backbone occurring naturally in, for example, animals and humans 
in mono-, di-, triglycerides. Therefore, this branching would not 
necessarily be seen as a foreign agent in the body. The POG has a 
preferred molecular weight in the same range as PEG. The structure for POG 
is shown in Knauf et al., 1988, J. Bio. Chem. 263:15064-15070, and a 
discussion of POG/protein conjugates is found in U.S. Pat. No. 4,766,106, 
both of which are hereby incorporated by reference in their entireties. 
After the liquid pharmaceutical composition is prepared, it is preferably 
lyophilized to prevent degradation and to preserve sterility. Methods for 
lyophilizing liquid compositions are known to those of ordinary skill in 
the art. Just prior to use, the composition may be reconstituted with a 
sterile diluent (Ringer's solution, distilled water, or sterile saline, 
for example) which may include additional ingredients. Upon 
reconstitution, the composition is preferably administered to subjects 
using those methods that are known to those skilled in the art. 
Administration to Affected Individuals 
Factor VIIa/TF/Xa binding proteins of the invention are use fill to treat 
mammals with sepsis or septic shock. Generally, conditions are 
characterized by high fever (&gt;38.5.degree. C.) or hypothermia 
(&gt;35.5.degree. C.), low blood pressure, tachypnea (&gt;20 breaths/minute), 
tachycardia (&gt;100 beats/minute), leukocytosis (&gt;15,000 cells/mm.sup.3) and 
thrombocytopenia (&lt;100,000 platelets/mm3). The factor VIIa/TF/Xa binding 
proteins of the invention are preferably administered as soon as the 
subject is suspected of being septic; presenting a &gt;20% drop in fibrinogen 
or appearance of fibrin split products, a rise in the subject's 
temperature and the diagnosis of leukopenia and hypotension associated 
with septic shock. As stated above, intravenous administration is 
preferred. Generally, factor VIIa/TF/Xa binding proteins of the invention 
are given at a dose between 1 .mu.g/kg and 20 mg/kg, more preferably 
between 20 .mu.g/kg and 10 mg/kg, most preferably between 1 and 7 mg/kg. 
Preferably, it is given as a bolus dose, to increase circulating levels by 
10-20 fold and for 4-6 hours after the bolus dose. Continuous infusion may 
also be used after the bolus dose. If so, the factor VIIa/TF/Xa binding 
proteins of the invention may be infused at a dose between 5 and 20 
.mu.g/kg/minute, more preferably between 7 and 15 .mu.g/kg/minute. 
The factor VIIa/TF/Xa binding proteins of the invention may be given in 
combination with other agents which would be effective to treat septic 
shock. For example, the following may be administered in combination with 
the factor VIIa/TF/Xa binding proteins of the invention: antibiotics that 
can treat the underlying bacterial infection; monoclonal antibodies that 
am directed against bacterial cell wall components; monoclonal antibodies 
and soluble receptors that can complex with cytokines that are involved in 
the sepsis pathway, including, but not limited to tumor necrosis factor 
(TNF), Interleukin-1, .gamma.-interferon and interleukin-8; and generally 
any agent or protein that can interact with cytokines or complement 
proteins in the sepsis pathway to reduce their effects and to attenuate 
sepsis or septic shock. 
Antibiotics useful in the present invention include those in the general 
category of: beta-lactam rings (penicillin), amino sugars in glycosidic 
linkage (amino glycosides), macrocyclic lactone rings (macrolides), 
polycyclic derivatives of napthacenecarboxamide (tetracyclines), 
nitrobenzene derivatives of dichloroacetic acid, peptides (bacitracin, 
gramicidin, and polymyxin), large rings with a conjugated double bond 
system (polyenes), sulfa drugs derived from sulfanilamide (sulfonamides), 
5-nitro-2-furanyl groups (nitrofurans), quinolone carboxylic acids 
(nalidixic acid), and many others. Other antibiotics and more versions of 
the above specific antibiotics may be found in Encyclopedia of Chemical 
Technology, 3rd Edition, Kirk-Othmer (ed.), Vol. 2, pages 782-1036 (1978) 
and Vol. 3, pages 1-78, Zinsser, MicroBiology, 17th Edition W. Joklik et 
al (Eds.) pages 235-277 (1980), or Dorland's Illustrated Medical 
Dictionary, 27th Edition, W.B. Saunders Company (1988). 
Other agents which may be combined with the factor VIIa/TF/Xa binding 
proteins of the invention include monoclonal antibodies directed to 
cytokines involved in the sepsis pathway, such as those monoclonal 
antibodies directed to IL-6 or M-CSF, such as shown in PCT US90/07411; 
monoclonal antibodies directed to TNF, such as shown in U.S. Pat. No. 
4,603,106; inhibitors of proteins that cleave the mature TNF prohormone 
from the cell in which it was produced, such as shown in PCT US90/03266 
and PCT US93/06120; antagonists of IL-1, such as shown in PCT US91/02460; 
inhibitors of IL-6 cytokine action such as activin, such as shown in PCT 
US90/00321; and receptor based inhibitors of various cytokine such as 
IL-1. Antibodies to or small molecule inhibitors of complement protein may 
also be employed. 
Generally, the factor VIIa/TF/Xa binding proteins of the invention may be 
useful for those diseases that occur due to the up-regulation of tissue 
factor brought: on by injury, trauma, endotoxin, TNF, cancer, IL-1 or 
other agents or conditions. 
EXAMPLES 
The present invention will now be illustrated by reference to the following 
examples which set forth particularly advantageous embodiments. However, 
it should be noted that these embodiments are illustrative and are not to 
be construed as restricting the invention in any way. 
EXAMPLE 1 
The shuttle vector pBS24 is described in Barr et al, EXPRESSION SYSTEMS & 
PROCESSES FOR rDNA PRODUCTS (American Chemical Society, 1991), pp 51-64). 
pBS24Ub is a derivative of pBS24.1, and contains an expression cassette 
flanked by unique Bam HI and Sal I restriction sites, the glucose 
regulatable ADH2/GAP promoter and a synthetic ubiquitin (Ub) gene. For 
construction of Ub fusions, a unique SstII site is generated in the 3' end 
of the Ub gene. The presence of the SstII site allows in-frame insertion 
of nucleotide sequences for expression as ubiquitin fusion peptides. 
Insertion can be accomplished by use of synthetic DNA adapters or PCR 
methodologies. Methods for using synthetic DNA adapters (linkers) are 
known in the art. All enzymatic modifications of DNA and protein are done 
according to the instructions provided by the manufacturer of the enzyme. 
PCR protocols are described in PCR PROTOCOLS: A GUIDE TO METHODS AND 
APPLICATIONS, (eds.) Innis, Gelfand, Sninsky and White (Academic Press, 
1990). In either case, the 5'-junction sequence will be: 
##STR1## 
and the 3' cloning site (Sal I) should be as close as possible to the 3' 
end of the termination codon. 
PCR was used to construct the ubiquitin/TFPI gene fusion in the 15.4 kb 
plasmid pLACI 4.1 shown in FIG. 1. TFPI encoding nucleic acid was 
amplified using standard PCR procedures with the primers SEQ ID NO: 21 and 
SEQ ID NO: 22. SEQ ID NO: 21 hybridizes to the 10 nucleotides at the 5' 
end of nucleic acid mature encoding TFPI and also contains ubiquitin 
sequence with the Sst II restriction site. SEQ ID NO: 22 hybridizes to the 
15 nucleotides at the 3' end of nucleic acid encoding mature TFPI and also 
trailing sequence with a Sal I restriction site. The sequences of these 
primers are as follows: 
__________________________________________________________________________ 
SEQ ID NO: 21 
GCTCCGCGGTGGCGATTCTGAGGAGGAGATGAAGAAC 
SEQ ID NO: 22 
TCTGTCGACTCACATATTTTTAACAAAAATTTCTTCAT 
__________________________________________________________________________ 
After amplification, the PCR product was digested with Sal I and Sst II 
using conditions specified by the manufacturer of the enzymes. The 
digested PCR product was then cloned into pBS24Ub, as described above, to 
produce pLACI 4.1. S. cerevisiae strain AB122 transformed with pLACI4.1 
has was deposited with the ATCC on Jul. 19, 1994 and has been given 
Accession Number 74291. 
pLACI 4.1 was used to transform three strains of Saccharomyces cerevisiae: 
VH6 (MAT .alpha., cir.degree., leu-2-112,-3, ura3, FoA, pep4::His3), AB122 
(MAT .alpha., cir.degree., leu2, ura3-52, prb1-1112, pep4-3, prc1-407) and 
JSC310 (as AB122, +ADR1 overexpression). Transformants of VH6 produced 
TFPI at levels of approximately 5% of total protein, transformants of 
AB122 produced TFPI at levels of approximately 10% of total protein and 
transformants of JSC310 produced TFPI at levels of approximately 15% of 
total protein. TFPI expressed according to this method was shown to have 
biological activity, that is TFPI showed both factor VIIa/TF and factor Xa 
inhibition. 
EXAMPLE 2 
TFPI was expressed as a full length fusion protein with prepro .alpha. 
factor leader. The .alpha.-factor/TFPI fusion protein was constructed 
using pAB125 as an intermediate plasmid. (Chang et al, J. Immunol. 
149:548-555 (1992)). pAB125 contains an expression cassette flanked by 
unique Bam HI and Sal I restriction sites, the glucose regulatable 
ADH2/GAP promoter and the .alpha.-factor prepro header sequence and 
processing site. For construction of .alpha.-factor fusions, a unique Xba 
I site is generated in the 3' end of the a factor leader gene sequence. 
The presence of the Xba I site allows in-frame insertion of nucleotide 
sequences for expression as a factor fusion peptides. Insertion can be 
accomplished by use of synthetic DNA adapters or PCR methodologies. In 
either case, the 5' junction sequence will be 
##STR2## 
and the 3' cloning site (Sal I) should be as close as possible to the 3' 
end of the termination codon. 
PCR was used to construct the .alpha.-factor/TFPI gene fusion in the 15.4 
kb plasmid pLACI2.1. TFPI encoding nucleic acid was amplified using 
standard PCR procedures with the primers SEQ ID NO: 24 and SEQ ID NO: 22. 
SEQ ID NO: 24 hybridizes to the 22 nucleotides at the 5' end of nucleic 
acid encoding mature TFPI and also contains .alpha.-factor sequence with 
the Xba I restriction site as shown above. 22 hybridizes to the 29 
nucleotides at the 3' end of nucleic acid encoding mature TFPI and 
trailing sequence with a Sal I restriction site. The sequence of SEQ ID 
NO: 24 is as follows: 
__________________________________________________________________________ 
SEQ ID NO: 24 
ATCTCTAGATAAAAGAGATTCTGAGGAAGATGAAGAAC 
__________________________________________________________________________ 
After amplification, the PCR product was digested with Sal I and Xba I 
using conditions specified by the manufacturer of the enzymes. The 
digested PCR product was then cloned into pAB125 which had been previously 
digested with Sal I and Xba I. The Barn HI to Sal I fragment of pAB125 
containing the .alpha.-factor/TFPI fusion protein was isolated and 
subcloned into pBS24.1 to produce pLACI 2.1 
pLACI 2.1 was used to transform the VH6 and AB122 strains of S. cerevisiae. 
EXAMPLE 3 
Truncated TFPI, containing amino acids 1-161 of mature TFPI, was expressed 
as an .alpha.-factor fusion protein for secretion. Preparation of 
TFPI-encoding sequence was accomplished essentially as in Example 2 using 
SEQ ID NO: 24 and a second primer, SEQ ID NO: 25, which hybridizes within 
the TFPI coding sequence. The sequence of SEQ ID NO: 25 is as follows: 
__________________________________________________________________________ 
SEQ ID NO: 25 
TCTGTCGACTCAGGTTCCATAATTATCCACCT 
__________________________________________________________________________ 
Alternatively, truncated TFPI can be expressed as a ubiquitin fusion 
protein and recovered from within the yeast cell. In order to prepare the 
appropriate coding sequence, the LACI4 and LACI2 primers are used to 
prepare the coding region for subcloning into pBS24-Ub as described above. 
EXAMPLE 4 
Yeast shake flask cultures were harvested by centrifugation and the 
supernatant fluids containing .alpha.-factor-TFPI were collected and 
filtered through 0.8.mu. membranes. The cells from TFPI cultures were 
pelleted by centrifugation. Cell lysates from .alpha.-factor-TFPI and 
ubiquitin-TFPI were prepared by disrupting cell pellets in 50 mM Tris pH 
7.5, 2 mM EDTA, 50 mM NaCl, 1 mM PMSF by vortexing with glass beads. In 
some cases, the cell lysates were further fractionated into soluble and 
insoluble fractions by centrifugation at 12000 g for 10 minutes at 
4.degree. C. 
Expression of TFPI was detected by SDS/PAGE followed by Coomassie staining 
or by Western blotting with a rabbit antiserum raised against the first 15 
amino acids of TFPI (NTP sera). Analysis of the soluble and insoluble 
fractions of cell lysates is shown in FIGS. 2A and 2B. Coomassie-stained 
gels (top panel) reveal the presence of unique bands migrating at 
.about.21 kD in the soluble fraction of .alpha.-factor-truncated TFPI (aa 
1-161) (Lane 2, FIG. 2A and FIG. 2B) and insoluble fractions of 
ubiquitin-truncated TFPI (aa 1-161) (Lane 5, FIG. 2A and FIG. 2B) which 
were verified to represent truncated TFPI by Western blotting (Lanes 2 and 
5, FIG. 2B). Full-length TFPI migrating at 35 kD was detectable in the 
insoluble fraction of ubiquitin-TFPI lysates (Lane 6, FIG. 2A and FIG. 2B) 
by Coomassie staining and Western blotting and was identified in the 
soluble fraction of .alpha.-factor TFPI lysates by Western blotting. (Lane 
3, FIG. 2B). Lanes 1 and 4 of FIGS. 2A and 2B contained negative contol 
proteins isolated as above from non-transformed yeast cell culture. 
Expression of secreted TFPI was detected by analysis of culture supernates. 
Biochemical analysis of protein expression included SDS/PAGE followed by 
Coomassie staining or Western blotting with NTP sera. Since truncated TFPI 
may be N-glycosylated at one site and full-length TFPI has the potential 
for modification by N-glycosylation at three sites, the supernatant 
samples were concentrated five-fold, dialyzed against Tris buffer and 
deglycosylated with N-glycanase according to the manufacturer's 
recommendations (Genzyme). As shown in FIG. 3, truncated TFPI was easily 
detectable by Coomassie staining and by Western blotting as major bands 
migrating at .about.21 and 25 kD (Lane 2, FIG. 3A and FIG. 3B). The 25 kD 
band was verified as representing glycosylated truncated TFPI by 
conversion of the doublet to the single 21 kD band following N-glycanase 
digestion (Lane 5, FIG. 3A and FIG. 3B). While secreted full-length TFPI 
was not as readily identified prior to N-glycanase treatment (Lane 3, FIG. 
3A and FIG. 3B), the 35 kD TFPI band could be observed following 
deglycosylation (Lane 6, FIG. 3A and FIG. 3B). Lanes 1 and 4 of FIGS. 3A 
and 3B contained negative contol proteins isolated as above from 
non-transformed yeast cell culture. 
Biological assays were performed using supernatants and soluble fractions 
of cell lysates of TFPI cultures. A prothrombin time clotting assay (Wun 
et al, J. Biol. Chem. 265:16096 (1990)) indicated weak TFPI activity in 
unconcentrated supernatants from cultures secreting truncated TFPI, while 
the soluble fraction of intracellularly expressed truncated and 
full-length TFPI exhibited TFPI activity as compared to the intracellular 
control. A more sensitive assay of TFPI activity, the inhibition of factor 
Xa activity (Wun et al, ibid) was performed on supernates from cultures 
producing truncated and full-length TFPI. As shown in FIG. 4, significant 
Xa inhibitory activity was evident in supernates from cultures secreting 
truncated TFPI (L161) and full length TFPI (L-FL) as compared to negative 
control (V2) yeast culture. 
EXAMPLE 5 
Potential sites for N-linked glycosylation within TFPI are removed using 
overlapping PCR as described in Innis et al, supra. As in the previous 
examples, TFPI-encoding sequences are prepared by PCR, cloned in pAB125 
and further subcloned in pBS24.1 for expression. 
For replacement of the asparagine at position 116 with glutamine, the 
following overlapping primers are used: 
__________________________________________________________________________ 
SEQ ID NO: 26 
AGGTATTTTTATAACAATCAGACAAAACAGTGT 
SEQ ID NO: 27 
GAAACGTTCACACTGTTTTGTCTGATTGTTATA 
__________________________________________________________________________ 
For replacement of the asparagine at position 167 with glutamine, the 
following overlapping primers are used: 
__________________________________________________________________________ 
SEQ ID NO: 28 
CCAGCTCAATGCTGTGAATAACTCCCTGACTCCG 
SEQ ID NO: 29 
CTTGGTTGATTGCGGAGTCAGGGAGTTATTCACAGC 
__________________________________________________________________________ 
For replacement of the asparagine at position 227 with glutamine, the 
following overlapping primers are used: 
__________________________________________________________________________ 
SEQ ID NO: 30 
GGGGGAAATGAAAACAATTTTACTTCCAAACAA 
SEQ ID NO: 31 
CCTCAGACATTCTTGTTTGGAAGTAAAATTGTTTTC 
__________________________________________________________________________ 
In each case, the LACI1 and LACI3 primers are used to provide the necessary 
Xba I and Sal I sites for cloning. To introduce more than one mutation, 
thereby eliminating another potential glycosylation site, sequential 
rounds of overlapping PCR may be used. 
EXAMPLE 6 
In order to produce a mutein of TFPI containing a Lys to Arg substitution 
in the P1 reactive site of Kunitz-type domain 1, a Bgl II-Sal I fragment 
from pBS24Ub/TFPI1 was subcloned into pSP72 (commercially available from 
Promega). A Ava III-Bsp HI fragment was then prepared using the following 
primers: 
______________________________________ 
SEQ ID NO: 32 
CCGATGCATTCATTTTGTGCATTC 
SEQ ID NO: 33 
CCTCATGATTGCCCGACATGGGCC 
______________________________________ 
The second primer contains a single mismatch from the native sequence 
resulting in the desired Lys to Arg mutation in the resulting fragment. 
This mutated sequence is then used to replace the Ava III-Bsp HI fragment 
in the TFPI-encoding sequence previously cloned into pSP72. The resulting 
plasmid is then digested with Sal II and Sal I and cloned into pBS24-Ub 
for expression. 
EXAMPLE 7 
Constructs encoding chimeric TFPI proteins in which the Kunitz-type domain 
1 of TFPI is replaced with the Kunitz-type domain 1 of TFPI-2 were 
prepared using overlapping PCR. A Sst II-Bsm I restriction fragment, 
containing Kunitz-type domain 1 of TFPI-2 and part of Kunitz-type domain 2 
of TFPI, was generated by overlapping PCR. The primers used were: 
__________________________________________________________________________ 
SEQ ID NO: 34 
GGTCCGCGGTGGTGATGCTGCTCAGGAGC 
SEQ ID NO: 35 
GCAATGTTGTTTTTTCTATCCTCCAGCAAGCAT 
__________________________________________________________________________ 
These two primers were used to prepare a 93 bp fragment using the TFPI-2 
coding sequence as a template. The TFPI-2 coding sequence was cloned using 
PCR. Primers for cloning were derived from the sequence published in 
Sprecher et al, Proc. Nat. Acad. Sci. U.S.A. 91:3353-3357 (1994). 
A 54 bp fragment was then prepared using TFPI coding sequence as a template 
and the following primers: 
__________________________________________________________________________ 
SEQ ID NO: 36 
GGATAGAAAAAAACAACATTGCAACAAGAAAAGC 
SEQ ID NO: 37 
GGTTCTTGCATTCTTCCAGTGTCTCAAAATTG 
__________________________________________________________________________ 
Overlapping PCR was then performed to join these two fragments using the 
primers TFOLA and TFOLD. The 126 bp product was then digested with Sst II 
and Bsm I and exchanged for the equivalent Sst I-Bsm I fragment in 
pSP72/TFP1. This construct was subsequently digested with Sst II and Sal I 
and the resulting fragment cloned in pBS24-Ub for expression. 
EXAMPLE 8 
Yeast strains are produced containing och1, mnn1 and alg3 mutations 
resulting in reduction of carbohydrate attached to secreted TFPI molecules 
of the invention. The genes are cloned using standard PCR techniques based 
on sequences available to one skilled in the art. 
Deletions of 300 bp or more in the OCH1, MNN1 and ALG3 genes are introduced 
into the coding region of each of these genes. The genes with sequences 
deleted are then cloned into URA3-based integrating vector. The wild type 
genes for each of the three are sequentially replaced using the 
pop-in/pop-out replacement vector as described in Scherer and Davis, Proc. 
Nat. Acad. Sci. U.S.A. 76:4951 (1979). 
The foregoing discussion and examples only illustrate the present 
invention, persons of ordinary skill in the art will appreciate that the 
invention can be implemented in other ways, and the invention is defined 
solely by reference to the claims. Further, all references, patents and 
patent applications cited in the foregoing specification are incorporated 
herein by reference. 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 37 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 51 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
CysAlaPheLysAlaAspAspGlyProCysLysAlaIleMetLysArg 
151015 
PhePhePheAsnIlePheThrArgGlnCysGluGluPheIleTyrGly 
202530 
GlyCysGluGlyAsnGlnAsnArgPheGluSerLeuGluGluCysLys 
354045 
LysMetCys 
50 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 51 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
CysLeuLeuProLeuAspTyrGlyProCysArgAlaLeuLeuLeuArg 
151015 
TyrTyrTyrAspArgTyrThrGlnSerCysArgGlnPheLeuTyrGly 
202530 
GlyCysGluGlyAsnAlaAsnAsnPheTyrThrTrpGluAlaCysAsp 
354045 
AspAlaCys 
50 
(2) INFORMATION FOR SEQ ID NO:3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 51 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
CysPheLeuGluGluAspProGlyIleCysArgGlyTyrIleThrArg 
151015 
TyrPheTyrAsnAsnGlnThrLysGlnCysGluArgPheLysTyrGly 
202530 
GlyCysLeuGlyAsnMetAsnAsnPheGluThrLeuGluGluCysLys 
354045 
AsnIleCys 
50 
(2) INFORMATION FOR SEQ ID NO:4: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 54 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
CysArgLeuGlnValSerValAspAspGlnCysGluGlySerThrGlu 
151015 
LysTyrPhePheAsnLeuSerSerMetThrCysGluLysPhePheSer 
202530 
GlyGlyCysHisArgAsnArgIleGluAsnArgPheProAspGluAla 
354045 
ThrCysMetGlyPheCys 
50 
(2) INFORMATION FOR SEQ ID NO:5: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 51 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
CysLeuThrProAlaAspArgGlyLeuCysArgAlaAsnGluAsnArg 
151015 
PheTyrTyrAsnSerValIleGlyLysCysArgProPheLysTyrSer 
202530 
GlyCysGlyGlyAsnGluAsnAsnPheThrSerLysGlnGluCysLeu 
354045 
ArgAlaCys 
50 
(2) INFORMATION FOR SEQ ID NO:6: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 51 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: 
CysTyrSerProLysAspGluGlyLeuCysSerAlaAsnValThrArg 
151015 
TyrTyrPheAsnProArgTyrArgThrCysAspAlaPheThrTyrThr 
202530 
GlyCysGlyGlyAsnAspAsnAsnPheValSerArgGluAspCysLys 
354045 
ArgAlaCys 
50 
(2) INFORMATION FOR SEQ ID NO:7: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 37 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: 
LysLysGlyPheIleGlnArgIleSerLysGlyGlyLeuIleLysThr 
151015 
LysArgLysArgLysLysGlnArgValLysIleAlaTyrGluGluIle 
202530 
PheValLysAsnMet 
35 
(2) INFORMATION FOR SEQ ID NO:8: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 27 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: 
AlaLysAlaLeuLysLysLysLysLysMetProLysLeuArgPheAla 
151015 
SerArgIleArgLysIleArgLysLysGlnPhe 
2025 
(2) INFORMATION FOR SEQ ID NO:9: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 276 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: 
AspSerGluGluAspGluGluHisThrIleIleThrAspThrGluLeu 
151015 
ProProLeuLysLeuMetHisSerPheCysAlaPheLysAlaAspAsp 
202530 
GlyProCysArgAlaIleMetLysArgPhePhePheAsnIlePheThr 
354045 
ArgGlnCysGluGluPheIleTyrGlyGlyCysGluGlyAsnGlnAsn 
505560 
ArgPheGluSerLeuGluGluCysLysLysMetCysThrArgAspAsn 
65707580 
AlaAsnArgIleIleLysThrThrLeuGlnGlnGluLysProAspPhe 
859095 
CysPheLeuGluGluAspProGlyIleCysArgGlyTyrIleThrArg 
100105110 
TyrPheTyrAsnAsnGlnThrLysGlnCysGluArgPheLysTyrGly 
115120125 
GlyCysLeuGlyAsnMetAsnAsnPheGluThrLeuGluGluCysLys 
130135140 
AsnIleCysGluAspGlyProAsnGlyPheGlnValAspAsnTyrGly 
145150155160 
ThrGlnLeuAsnAlaValAsnAsnSerLeuThrProGlnSerThrLys 
165170175 
ValProSerLeuPheGluPheHisGlyProSerTrpCysLeuThrPro 
180185190 
AlaAspArgGlyLeuCysArgAlaAsnGluAsnArgPheTyrTyrAsn 
195200205 
SerValIleGlyLysCysArgProPheLysTyrSerGlyCysGlyGly 
210215220 
AsnGluAsnAsnPheThrSerLysGlnGluCysLeuArgAlaCysLys 
225230235240 
LysGlyPheIleGlnArgIleSerLysGlyGlyLeuIleLysThrLys 
245250255 
ArgLysArgLysLysGlnArgValLysIleAlaTyrGluGluIlePhe 
260265270 
ValLysAsnMet 
275 
(2) INFORMATION FOR SEQ ID NO:10: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 23 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: 
LysThrLysArgLysArgLysLysGlnArgValLysIleAlaTyrGlu 
151015 
GluIlePheValLysAsnMet 
20 
(2) INFORMATION FOR SEQ ID NO:11: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 23 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: 
LysLysLysLysLysMetProLysLeuArgPheAlaSerArgIleArg 
151015 
LysIleArgLysLysGlnPhe 
20 
(2) INFORMATION FOR SEQ ID NO:12: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 17 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: 
AlaLysLeuAsnCysArgLeuTyrArgLysAlaAsnLysSerSerLys 
151015 
Leu 
(2) INFORMATION FOR SEQ ID NO:13: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 15 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: 
ThrSerAspGlnIleHisPhePhePheAlaLysLeuAsnCysArg 
151015 
(2) INFORMATION FOR SEQ ID NO:14: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: 
SerGluLysThrLeuArgLysTrpLeuLysMetPheLysLysArgGlu 
151015 
LeuGluGluTyr 
20 
(2) INFORMATION FOR SEQ ID NO:15: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 15 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15: 
HisArgHisHisProArgGluMetLysLysArgValGluAspLeu 
151015 
(2) INFORMATION FOR SEQ ID NO:16: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 18 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: 
PheArgLysLeuThrHisArgLeuPheArgArgAsnPheGlyTyrThr 
151015 
LeuArg 
(2) INFORMATION FOR SEQ ID NO:17: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 12 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: 
LeuTyrLysLysIleLeuLysLysLeuLeuGluAla 
1510 
(2) INFORMATION FOR SEQ ID NO:18: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 26 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: 
AsnGlyLeuLysArgAspLysLeuGlyCysGluTyrCysGluCysArg 
151015 
ProLysArgLysLeuIleProArgLeuSer 
2025 
(2) INFORMATION FOR SEQ ID NO:19: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 161 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: 
AspSerGluGluAspGluGluHisThrIleIleThrAspThrGluLeu 
151015 
ProProLeuLysLeuMetHisSerPheCysAlaPheLysAlaAspAsp 
202530 
GlyProCysArgAlaIleMetLysArgPhePhePheAsnIlePheThr 
354045 
ArgGlnCysGluGluPheIleTyrGlyGlyCysGluGlyAsnGlnAsn 
505560 
ArgPheGluSerLeuGluGluCysLysLysMetCysThrArgAspAsn 
65707580 
AlaAsnArgIleIleLysThrThrLeuGlnGlnGluLysProAspPhe 
859095 
CysPheLeuGluGluAspProGlyIleCysArgGlyTyrIleThrArg 
100105110 
TyrPheTyrAsnGlnGlnThrLysGlnCysGluArgPheLysTyrGly 
115120125 
GlyCysLeuGlyAsnMetAsnAsnPheGluThrLeuGluGluCysLys 
130135140 
AsnIleCysGluAspGlyProAsnGlyPheGlnValAspAsnTyrGly 
145150155160 
Thr 
(2) INFORMATION FOR SEQ ID NO:20: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 9 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc = "Adapter" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20: 
CCGCGGGGC9 
(2) INFORMATION FOR SEQ ID NO:21: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 37 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc = "primer" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: 
GCTCCGCGGTGGCGATTCTGAGGAGGAGATGAAGAAC37 
(2) INFORMATION FOR SEQ ID NO:22: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 38 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc = "primer" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: 
TCTGTCGACTCACATATTTTTAACAAAAATTTCTTCAT38 
(2) INFORMATION FOR SEQ ID NO:23: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 13 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc = "adapter" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: 
TCTAGATAAAAGA13 
(2) INFORMATION FOR SEQ ID NO:24: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 38 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc = "primer" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24: 
ATCTCTAGATAAAAGAGATTCTGAGGAAGATGAAGAAC38 
(2) INFORMATION FOR SEQ ID NO:25: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 32 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc = "primer" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25: 
TCTGTCGACTCAGGTTCCATAATTATCCACCT32 
(2) INFORMATION FOR SEQ ID NO:26: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 33 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc = "primer" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26: 
AGGTATTTTTATAACAATCAGACAAAACAGTGT33 
(2) INFORMATION FOR SEQ ID NO:27: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 33 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc = "primer" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:27: 
GAAACGTTCACACTGTTTTGTCTGATTGTTATA33 
(2) INFORMATION FOR SEQ ID NO:28: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 34 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc = "primer" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:28: 
CCAGCTCAATGCTGTGAATAACTCCCTGACTCCG34 
(2) INFORMATION FOR SEQ ID NO:29: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 36 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc = "primer" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:29: 
CTTGGTTGATTGCGGAGTCAGGGAGTTATTCACAGC36 
(2) INFORMATION FOR SEQ ID NO:30: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 33 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc = "primer" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:30: 
GGGGGAAATGAAAACAATTTTACTTCCAAACAA33 
(2) INFORMATION FOR SEQ ID NO:31: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 36 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc = "primer" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:31: 
CCTCAGACATTCTTGTTTGGAAGTAAAATTGTTTTC36 
(2) INFORMATION FOR SEQ ID NO:32: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 24 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc = "primer" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:32: 
CCGATGCATTCATTTTGTGCATTC24 
(2) INFORMATION FOR SEQ ID NO:33: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 24 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc = "primer" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:33: 
CCTCATGATTGCCCGACATGGGCC24 
(2) INFORMATION FOR SEQ ID NO:34: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 29 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc = "primer" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:34: 
GGTCCGCGGTGGTGATGCTGCTCAGGAGC29 
(2) INFORMATION FOR SEQ ID NO:35: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 33 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc = "primer" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:35: 
GCAATGTTGTTTTTTCTATCCTCCAGCAAGCAT33 
(2) INFORMATION FOR SEQ ID NO:36: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 34 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc = "primer" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:36: 
GGATAGAAAAAAACAACATTGCAACAAGAAAAGC34 
(2) INFORMATION FOR SEQ ID NO:37: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 32 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc = "primer" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:37: 
GGTTCTTGCATTCTTCCAGTGTCTCAAAATTG32 
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