U.S. patent application number 11/092336 was filed with the patent office on 2005-09-01 for nematode-extracted serine protease inhibitors and anticoagulant proteins.
This patent application is currently assigned to Corvas International, Inc., a Delaware corporation. Invention is credited to Bergum, Peter W., Gansemans, Yannick Georges Jozef, Jespers, Laurent Stephane, LaRoche, Yves Rene, Lauwereys, Marc Josef, Messens, Joris Hilda Lieven, Moyle, Matthew, Stanssens, Patrick Eric Hugo, Vlasuk, George Philip.
Application Number | 20050191724 11/092336 |
Document ID | / |
Family ID | 27541063 |
Filed Date | 2005-09-01 |
United States Patent
Application |
20050191724 |
Kind Code |
A1 |
Vlasuk, George Philip ; et
al. |
September 1, 2005 |
Nematode-extracted serine protease inhibitors and anticoagulant
proteins
Abstract
Proteins which have activity as anticoagulants and/or serine
protease inhibitors and have at least one NAP domain and are
described. Certain of these proteins have factor Xa inhibitory
activity and others have activity as inhibitors of factor VIIa/TF.
These proteins can be isolated from natural sources as nematodes,
chemically synthesized or made by recombinant methods using various
DNA expression systems.
Inventors: |
Vlasuk, George Philip;
(Carlsbad, CA) ; Stanssens, Patrick Eric Hugo;
(St-Martens-Latem, BE) ; Messens, Joris Hilda Lieven;
(Dilbeek, BE) ; Lauwereys, Marc Josef; (Haabert,
BE) ; LaRoche, Yves Rene; (Bruxelles, BE) ;
Jespers, Laurent Stephane; (Tervuren, BE) ;
Gansemans, Yannick Georges Jozef; (Ichtegem, BE) ;
Moyle, Matthew; (Thousand Oaks, CA) ; Bergum, Peter
W.; (San Diego, CA) |
Correspondence
Address: |
FISH & RICHARDSON PC
225 FRANKLIN ST
BOSTON
MA
02110
US
|
Assignee: |
Corvas International, Inc., a
Delaware corporation
|
Family ID: |
27541063 |
Appl. No.: |
11/092336 |
Filed: |
March 29, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11092336 |
Mar 29, 2005 |
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09498556 |
Feb 4, 2000 |
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6872808 |
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09498556 |
Feb 4, 2000 |
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08809455 |
Nov 24, 1997 |
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6090916 |
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08809455 |
Nov 24, 1997 |
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PCT/US95/13231 |
Oct 17, 1995 |
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08809455 |
Nov 24, 1997 |
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08461965 |
Jun 5, 1995 |
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5872098 |
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08809455 |
Nov 24, 1997 |
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08465380 |
Jun 5, 1995 |
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5863894 |
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08809455 |
Nov 24, 1997 |
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08486397 |
Jun 5, 1995 |
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5866542 |
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08809455 |
Nov 24, 1997 |
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08486399 |
Jun 5, 1995 |
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5866543 |
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08809455 |
Nov 24, 1997 |
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08326110 |
Oct 18, 1994 |
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5945275 |
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Current U.S.
Class: |
435/69.1 ;
435/320.1; 435/325; 514/14.3; 514/14.5; 514/20.3; 514/4.6; 530/350;
536/23.2 |
Current CPC
Class: |
C07K 14/811 20130101;
Y10S 530/829 20130101; C12P 21/02 20130101; A61P 43/00 20180101;
A61P 7/02 20180101; A61K 38/55 20130101 |
Class at
Publication: |
435/069.1 ;
536/023.2; 435/320.1; 435/325; 530/350; 514/012 |
International
Class: |
C07K 014/435; C07H
021/04; A61K 038/17; C12P 021/06 |
Claims
1-269. (canceled)
270. A pharmaceutical composition comprising: (a) a polypeptide
having the amino acid sequence
LysAlaThrMetGlnCysGlyGluAsnGluLysTyrAspSerCysGlySerLy-
sGluCysAspLys
LysCysLysTyrAspGlyValGluGluGluAspAspGluGluProAsnValProCysLeu-
ValArg
ValCysHisGlnAspCysValCysGluGluGlyPheTyrArgAsnLysAspAspLysCysValSer
AlaGluAspCysGluLeuAspAsnMetAspPheIleTyrProGlyThrArgAsnPro
(rNAPc2/Pro); and (b) a pharmaceutically acceptable carrier.
271. A method for producing a polypeptide comprising the amino acid
sequence of rNAPc2, the method comprising (a) providing a cell
encoding a polypeptide comprising the amino acid sequence of
rNAPc2; (b) culturing the cell under conditions suitable for the
production of the polypeptide comprising the amino acid sequence of
rNAPc2; and (c) purifying the polypeptide from the cell culture
supernatant.
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] This application is a Continuation-in-Part of U.S. Ser. Nos.
08/461,965, 08/465,380, 08/486,397 and 08/486,399, all filed on
Jun. 5, 1995, each of which is a continuation-in-part of U.S. Ser.
No. 08/326,110, filed Oct. 18, 1995; the disclosures of all these
applications are incorporated herein by reference.
FIELD OF THE INVENTION
[0002] The present invention relates to specific proteins as well
as recombinant versions of these proteins which are serine protease
inhibitors, including potent anticoagulants in human plasma. These
proteins include certain proteins extracted from nematodes. In
another aspect, the present invention relates to compositions
comprising these proteins, which are useful as potent and specific
inhibitors of blood coagulation enzymes in vitro and in vivo, and
methods for their use as in vitro diagnostic agents, or as in vivo
therapeutic agents, to prevent the clotting of blood. In a further
aspect, the invention relates to nucleic acid sequences, including
mRNA and DNA, encoding the proteins and their use in vectors to
transfect or transform host cells and as probes to isolate certain
related genes in other species and organisms.
BACKGROUND AND INTRODUCTION TO THE INVENTION
[0003] Normal hemostasis is the result of a delicate balance
between the processes of clot formation (blood coagulation) and
clot dissolution (fibrinolysis). The complex interactions between
blood cells, specific plasma proteins and the vascular surface,
maintain the fluidity of blood unless injury occurs. Damage to the
endothelial barrier lining the vascular wall exposes underlying
tissue to these blood components. This in turn triggers a series of
biochemical reactions altering the hemostatic balance in favor of
blood coagulation which can either result in the desired formation
of a hemostatic plug stemming the loss of blood or the undesirable
formation of an occlusive intravascular thrombus resulting in
reduced or complete lack of blood flow to the affected organ.
[0004] The blood coagulation response is the culmination of a
series of amplified reactions in which several specific zymogens of
serine proteases in plasma are activated by limited proteolysis.
This series of reactions results in the formation of an insoluble
matrix composed of fibrin and cellular components which is required
for the stabilization of the primary hemostatic plug or thrombus.
The initiation and propagation of the proteolytic activation
reactions occurs through a series of amplified pathways which are
localized to membranous surfaces at the site of vascular injury
(Mann, K. G., Nesheim, M. E., Church, W. R., Haley, P. and
Krishnaswamy, S. (1990) Blood 76: 1-16. and Lawson, J. H.,
Kalafatis, M., Stram, S., and Mann, K. G. (1994) J. Biol. Chem.
269: 23357-23366).
[0005] Initiation of the blood coagulation response to vascular
injury follows the formation of a catalytic complex composed of
serine protease factor VIIa and the non-enzymatic co-factor, tissue
factor (TF) (Rappaport, S. I. and Rao, L. V. M. (1992)
Arteriosclerosis and Thrombosis 12: 1112-1121). This response
appears to be exclusively regulated by the exposure of
subendothelial TF to trace circulating levels of factor VIIa and
its zymogen factor VII, following a focal breakdown in vascular
integrity. Autoactivation results in an increase in the number of
factor VIIa/TF complexes which are responsible for the formation of
the serine protease factor Xa. It is believed that in addition to
the factor VIIa/TF complex, the small amount of factor Xa which is
formed primes the coagulation response through the proteolytic
modification of factor IX to factor IX.sub.alpha which in turn is
converted to the active serine protease factor IXa.sub.beta by the
factor VIIa/TF complex (Mann, K. G., Krishnaswamy, S. and Lawson,
J. H. (1992) Sem. Hematology 29: 213-226.). It is factor
IXa.sub.beta in complex with activated factor VIIIa, which appears
to be responsible for the production of significant quantities of
factor Xa which subsequently catalyzes the penultimate step in the
blood coagulation cascade; the formation of the serine protease
thrombin.
[0006] Factor Xa catalyzes the formation of thrombin following the
assembly of the prothrombinase complex which is composed of factor
Xa, the non-enzymatic co-factor Va and the substrate prothrombin
(factor II) assembled in most cases, on the surface of activated
platelets which are adhered at the site of injury (Fuster, V.,
Badimon, L., Badimon, J. J. and Chesebro, J. H. (1992) New Engl. J.
Med. 326: 310-318). In the arterial vasculature, the resulting
amplified "burst" of thrombin generation catalyzed by
prothrombinase causes a high level of this protease locally which
is responsible for the formation of fibrin and the further
recruitment of additional platelets as well as the covalent
stabilization of the clot through the activation of the
transglutaminase zymogen factor XIII. In addition, the coagulation
response is further propagated through the thrombin-mediated
proteolytic feedback activation of the non-enzymatic co-factors V
and VIII resulting in more prothrombinase formation and subsequent
thrombin generation (Hemker, H. C. and Kessels, H. (1991)
Haemostasis 21: 189-196).
[0007] Substances which interfere in the process of blood
coagulation (anticoagulants) have been demonstrated to be important
therapeutic agents in the treatment and prevention of thrombotic
disorders (Kessler, C. M. (1991) Chest 99: 97S-112S and Cairns, J.
A., Hirsh, J., Lewis, H. D., Resnekov, L., and Theroux, P. (1992)
Chest 102: 456S-481S). The currently approved clinical
anticoagulants have been associated with a number of adverse
effects owing to the relatively non-specific nature of their
effects on the blood coagulation cascade (Levine, M. N., Hirsh, J.,
Landefeld, S., and Raskob, G. (1992) Chest 102: 352S-363S). This
has stimulated the search for more effective anticoagulant agents
which can more effectively control the activity of the coagulation
cascade by selectively interfering with specific reactions in this
process which may have a positive effect in reducing the
complications of anticoagulant therapy (Weitz, J., and Hirsh, J.
(1993) J. Lab. Clin. Med. 122: 364-373). In another aspect, this
search has focused on normal human proteins which serve as
endogenous anticoagulants in controlling the activity of the blood
coagulation cascade. In addition, various hematophageous organisms
have been investigated because of their ability to effectively
anticoagulate the blood meal during and following feeding on their
hosts suggesting that they have evolved effective anticoagulant
strategies which may be useful as therapeutic agents.
[0008] A plasma protein, Tissue Factor Pathway Inhibitor (TFPI),
contains three consecutive Kunitz domains and has been reported to
inhibit the enzyme activity of factor Xa directly and, in a factor
Xa-dependent manner, inhibit the enzyme activity of the factor
VIIa-tissue factor complex. Salvensen, G., and Pizzo, S. V.,
"Proteinase Inhibitors: .alpha.-Macroglobulins, Serpins, and
Kunis", "Hemostasis and Thrombosis, Third Edition, pp. 251-253, J.
B. Lippincott Company (Edit. R. W. Colman et al. 1994). A cDNA
sequence encoding TFPI has been reported, and the cloned protein
was reported to have a molecular weight of 31,950 daltons and
contain 276 amino acids. Broze, G. J. and Girad, T. J., U.S. Pat.
No. 5,106,833, col. 1, (1992). Various recombinant proteins derived
from TFPI have been reported. Girad, T. J. and Broze, G. J., EP
439,442 (1991); Rasmussen, J. S. and Nordfand, O. J., WO 91/02753
(1991); and Broze, G. J. and Girad, T. J., U.S. Pat. No. 5,106,833,
col. 1, (1992).
[0009] Antistasin, a protein comprised of 119 amino acids and found
in the salivary gland of the Mexican leech, Haementeria
officinalis, has been reported to inhibit the enzyme activity of
factor Xa. Tuszynski et al., J. Biol. Chem, 262:9718 (1987); Nutt,
et al., J. Biol. Chem, 263:10162 (1988). A 6,000 daltons
recombinant protein containing 58 amino acids with a high degree
homology to antistasin's amino-terminus amino acids 1 through 58
has been reported to inhibit the enzyme activity of factor Xa.
Tung, J. et al., EP 454,372 (Oct. 30, 1991); Tung, J. et al., U.S.
Pat. No. 5,189,019 (Feb. 23, 1993).
[0010] Tick Anticoagulant Peptide (TAP), a protein comprised of 60
amino acids and isolated from the soft tick, Ornithodoros moubata,
has been reported to inhibit the enzyme activity of factor Xa but
not factor VIIa. Waxman, L. et al., Science, 248:593 (1990). TAP
made by recombinant methods has been reported. Vlausk, G. P. et
al., EP 419,099 (1991) and Vlausk, G. P. et al., U.S. Pat. No.
5,239,058 (1993).
[0011] The dog hookworm, Ancylostoma caninum, which can also infect
humans, has been reported to contain a potent anticoagulant
substance which inhibited coagulation of blood in vitro. Loeb, L.
and Smith, A. J., Proc. Pathol. Soc. Philadelphia, 7:173-187
(1904). Extracts of A. caninum were reported to prolong prothrombin
time and partial thromboplastin time in human plasma with the
anticoagulant effect being reported attributable to inhibition of
factor Xa but not thrombin. Spellman, Jr., J. J. and Nossel, H. L.,
Am. J. Physiol., 220:922-927 (1971). More recently, soluble protein
extracts of A. caninum were reported to prolong prothrombin time
and partial thromboplastin time in human plasma in vitro. The
anticoagulant effect was reported to be attributable to inhibition
of human factor Xa but not thrombin, Cappello, M, et al., J.
Infect. Diseases, 167:1474-1477 (1993), and to inhibition of factor
Xa and factor VIIa (WO94/25000; U.S. Pat. No. 5,427,937).
[0012] The human hookworm, Ancylostoma ceylanicum, has also been
reported to contain an anticoagulant. Extracts of A. ceylanicum
have been reported to prolong prothrombin time and partial
thromboplastin time in dog and human plasma in vitro. Carroll, S.
M., et al., Thromb. Haemostas. (Stuttgart), 51:222-227 (1984).
[0013] Soluble extracts of the non-hematophagous parasite, Ascaris
suum, have been reported to contain an anticoagulant. These
extracts were reported to prolong the clotting of whole blood, as
well as clotting time in the kaolin-activated partial
thromboplastin time test but not in the prothrombin time test.
Crawford, G. P. M. et al., J. Parasitol., 68: 1044-1047 (1982).
[0014] Chymotrypsin/elastase inhibitor-1 and its major isoforms,
trypsin inhibitor-1 and chymotrypsin/elastase inhibitor-4, isolated
from Ascaris suum, were reported to be serine protease inhibitors
and share a common pattern of five-disulfide bridges. Bernard, V.
D. and Peanasky, R. J., Arch. Biochem. Biophys., 303:367-376
(1993); Huang, K. et al., Structure, 2:679-689 (1994); and
Grasberger, B. L. et al., Structure, 2:669-678 (1994). There was no
indication that the reported serine protease inhibitors had
anticoagulant activity.
[0015] Secretions of the hookworm Necator americanus are reported
to prolong human plasma clotting times, inhibit the amidolytic
activity of human FXa using a fluorogenic substrate, inhibit
multiple agonist-induced platelet dense granule release, and
degrade fibrinogen. Pritchard, D. I. and B. Furmidge, Thromb.
Haemost. 73: 546 (1995) (WO95/12615).
SUMMARY OF THE INVENTION
[0016] The present invention is directed to isolated proteins
having serine protease inhibiting activity and/or anticoagulant
activity and including at least one NAP domain. We refer to these
proteins as Nematode-extracted Anticoagulant Proteins or "NAPs".
"NAP domain" refers to a sequence of the isolated protein, or NAP,
believed to have the inhibitory activity, as further defined herein
below. The anticoagulant activity of these proteins may be assessed
by their activities in increasing clotting time of human plasma in
the prothrombin time (PT) and activated partial thromboplastin time
(aPTT) assays, as well as by their ability to inhibit the blood
coagulation enzymes factor Xa or factor VIIa/TF. It is believed
that the NAP domain is responsible for the observed anticoagulant
activity of these proteins. Certain of these proteins have at least
one NAP domain which is an amino acid sequence containing less than
about 120 amino acid residues, and including 10 cysteine amino acid
residues.
[0017] In another aspect, the present invention is directed to a
method of preparing and isolating a cDNA molecule encoding a
protein exhibiting anticoagulant activity and having a NAP domain,
and to a recombinant cDNA molecule made by this method. This method
comprises the steps of:
[0018] (a) constructing a cDNA library from a species of nematode;
(b) ligating said cDNA library into an appropriate cloning vector;
(c) introducing said cloning vector containing said cDNA library
into an appropriate host cell; (d) contacting the cDNA molecules of
said host cell with a solution containing a hybridization probe
having a nucleic acid sequence comprising AAR GCi TAY CCi GAR TGY
GGi GAR AAY GAR TGG, [SEQ. ID. NO. 94] wherein R is A or G, Y is T
or C, and i is inosine; (e) detecting a recombinant cDNA molecule
which hybridizes to said probe; and (f) isolating said recombinant
cDNA molecule.
[0019] In another aspect, the present invention is directed to a
method of making a recombinant protein encoded by said cDNA which
has anticoagulant activity and which includes a NAP domain and to
recombinant proteins made by this method. This method comprises the
steps of: (a) constructing a cDNA library from a species of
nematode; (b) ligating said cDNA library into an appropriate
cloning vector; (c) introducing said cloning vector containing said
cDNA library into an appropriate host cell; (d) contacting the cDNA
molecules of said host cell with a solution containing a
hybridization probe having a nucleic acid sequence comprising AAR
GCi TAY CCi GAR TGY GGi GAR AAY GAR TGG, wherein R is A or G, Y is
T or C, and i is inosine [SEQ. ID. NO. 94]; (e) detecting a
recombinant cDNA molecule which hybridizes to said probe; (f)
isolating said recombinant cDNA molecule; (g) ligating the nucleic
acid sequence of said cDNA molecule which encodes said recombinant
protein into an appropriate expression cloning vector; (h)
transforming a second host cell with said expression cloning vector
containing said nucleic acid sequence of said cDNA molecule which
encodes said recombinant protein; (i) culturing the transformed
second host cell; and (j) isolating said recombinant protein
expressed by said second host cell. It is noted that when
describing production of recombinant proteins in certain expression
systems such as COS cells, the term "transfection" is
conventionally used in place of (and sometimes interchangeably
with) "transformation".
[0020] In another aspect, the present invention is directed to a
method of making a recombinant cDNA encoding a recombinant protein
having anticoagulant activity and having a NAP domain, comprising
the steps of: (a) isolating a cDNA library from a nematode; (b)
ligating said cDNA library into a cloning vector; (c) introducing
said cloning vector containing said cDNA library into a host cell;
(d) contacting the cDNA molecules of said host cells with a
solution comprising first and second hybridization probes, wherein
said first hybridization probe has the nucleic acid sequence
comprising AAG GCA TAC CCG GAG TGT GGT GAG AAT GAA TGG CTC GAC GAC
TGT GGA ACT CAG AAG CCA TGC GAG GCC AAG TGC AAT GAG GAA CCC CCT GAG
GAG GAA GAT CCG ATA TGC CGC TCA CGT GGT TGT TTA TTA CCT CCT GCT TGC
GTA TGC AAA GAC GGA TTC TAC AGA GAC ACG GTG ATC GGC GAC TGT GTT AGG
GAA GAA GAA TGC GAC CAA CAT GAG ATT ATA CAT GTC TGA [SEQ. ID. NO.
1], and said second hybridization probe has the nucleic acid
sequence comprising AAG GCA TAC CCG GAG TGT GGT GAG AAT GAA TGG CTC
GAC GTC TGT GGA ACT AAG AAG CCA TGC GAG GCC AAG TGC AGT GAG GAA GAG
GAG GAA GAT CCG ATA TGC CGA TCA TTT TCT TGT CCG GGT CCC GCT GCT TGC
GTA TGC GAA GAC GGA TTC TAC AGA GAC ACG GTG ATC GGC GAC TGT GTT AAG
GAA GAA GAA TGC GAC CAA CAT GAG ATT ATA CAT GTC TGA [SEQ. ID. NO.
2]; (e) detecting a recombinant cDNA molecule which hybridizes to
said mixture of said probes; and (f) isolating said recombinant
cDNA molecule.
[0021] In yet another aspect, the present invention is directed to
a method of making a recombinant cDNA encoding a protein having
anticoagulant activity and which encodes a NAP domain, comprising
the steps of: (a) isolating a cDNA library from a nematode; (b)
ligating said cDNA library into an appropriate phagemid expression
cloning vector; (c) transforming host cells with said vector
containing said cDNA library; (d) culturing said host cells; (e)
infecting said host cells with a helper phage; (f) separating phage
containing said cDNA library from said host cells; (g) combining a
solution of said phage containing said cDNA library with a solution
of biotinylated human factor Xa; (h) contacting a
streptavidin-coated solid phase with said solution containing said
phages containing said cDNA library, and said biotinylated human
factor Xa; (i) isolating phages which bind to said
streptavidin-coated solid phase; and (j) isolating the recombinant
cDNA molecule from phages which bind to said streptavidin-coated
solid phase.
[0022] In one preferred aspect, the present invention is directed
to a recombinant cDNA having a nucleic acid sequence selected from
the nucleic acid sequences depicted in FIG. 1, FIG. 3, FIGS. 7A to
7F, FIG. 9, FIGS. 13A to 13H, and FIG. 14.
[0023] The present invention also is directed to NAPs that inhibit
the catalytic activity of FXa, to NAPs that inhibit the catalytic
activity of the FVIIa/TF complex, and to NAPs that inhibit the
catalytic activity of a serine protease, as well as nucleic acids
encoding such NAPs and their methods of use.
DEFINITIONS
[0024] The term "amino acid" refers to the natural L-amino acids;
D-amino acids are included to the extent that a protein including
such D-amino acids retains biological activity. Natural L-amino
acids include alanine (Ala), arginine (Arg), asparagine (Asn),
aspartic acid (Asp), cysteine (Cys), glutamine (Gln), glutamic acid
(Glu), glycine (Gly), histidine (His), isoleucine (Ile), leucine
(Leu), lysine (Lys), methionine (Met), phenylalanine (Phe), proline
(Pro), serine (Ser), threonine (Thr), tryptophan (Trp), tyrosine
(Tyr) and valine (Val).
[0025] The term "amino acid residue" refers to radicals having the
structure: (1) --NH--CH(R)C(.dbd.O)--, wherein R is the
alpha-carbon side-chain group of an L-amino acid, except for
L-proline; or 1
[0026] for L-proline.
[0027] The term "peptide" refers to a sequence of amino acids
linked together through their alpha-amino and carboxylate groups by
peptide bonds. Such sequences as shown herein are presented in the
amino to carboxy direction, from left to right.
[0028] The term "protein" refers to a molecule comprised of one or
more peptides.
[0029] The term "cDNA" refers to complementary DNA.
[0030] The term "nucleic acid" refers to polymers in which bases
(e.g., purines or pyrimidines) are attached to a sugar phosphate
backbone. Nucleic acids include DNA and RNA.
[0031] The term "nucleic acid sequence" refers to the sequence of
nucleosides comprising a nucleic acid. Such sequences as shown
herein are presented in the 5' to 3' direction, from left to
right.
[0032] The term "recombinant DNA molecule" refers to a DNA molecule
created by ligating together pieces of DNA that are not normally
continguous.
[0033] The term "mRNA" refers to messenger ribonucleic acid.
[0034] The term "homology" refers to the degree of similarity of
DNA or peptide sequences.
[0035] The terms "Factor Xa" or "fXa" or "FXa" are synonymous and
are commonly known to mean a serine protease within the blood
coagulation cascade of enzymes that functions as part of the
prothrombinase complex to form the enzyme thrombin.
[0036] The phrase "Factor Xa inhibitory activity" means an activity
that inhibits the catalytic activity of fXa toward its
substrate.
[0037] The phrase "Factor Xa selective inhibitory activity" means
inhibitory activity that is selective toward Factor Xa compared to
other related enzymes, such as other serine proteases.
[0038] The phrase "Factor Xa inhibitor" is a compound having Factor
Xa inhibitory activity.
[0039] The terms "Factor VIIa/Tissue Factor" or "fVIIa/TF" or
"FVIIa/TF" are synonymous and are commonly known to mean a
catalytically active complex of the serine protease coagulation
factor VIIa (fVIIa) and the non-enzymatic protein Tissue Factor
(TF), wherein the complex is assembled on the surface of a
phospholipid membrane of defined composition.
[0040] The phrase "fVIIa/TF inhibitory activity" means an activity
that inhibits the catalytic activity of the fVIIa/TF complex in the
presence of fXa or catalytically inactive fXa derivative.
[0041] The phrase "fVIIa/TF selective inhibitory activity" means
fVIIa/TF inhibitory activity that is selective toward fVIIa/TF
compared to other related enzymes, such as other serine proteases,
including FVIIa and fXa.
[0042] The phrase a "fVIIa/TF inhibitor" is a compound having
fVIIa/TF inhibitory activity in the presence of fXa or
catalytically inactive fXa derivatives.
[0043] The phrase "serine protease" is commonly known to mean an
enzyme, comprising a triad of the amino acids histidine, aspartic
acid and serine, that catalytically cleaves an amide bond, wherein
the serine residue within the triad is involved in a covalent
manner in the catalytic cleavage. Serine proteases are rendered
catalytically inactive by covalent modification of the serine
residue within the catalytic triad by diisopropylfluorophosphate
(DFP).
[0044] The phrase "serine protease inhibitory activity" means an
activity that inhibits the catalytic activity of a serine
protease.
[0045] The phrase "serine protease selective inhibitory activity"
means inhibitory activity that is selective toward one serine
protease compared to other serine proteases.
[0046] The phrase "serine protease inhibitor" is a compound having
serine protease inhibitory activity.
[0047] The term "prothrombinase" is commonly known to mean a
catalytically active complex of the serine protease coagulation
Factor Xa (fXa) and the non-enzymatic protein Factor Va (fVa),
wherein the complex is assembled on the surface of a phospholipid
membrane of defined composition.
[0048] The phrase "anticoagulant activity" means an activity that
inhibits the clotting of blood, which includes the clotting of
plasma.
[0049] The term "selective", "selectivity", and permutations
thereof, when referring to NAP activity toward a certain enzyme,
mean the NAP inhibits the specified enzyme with at least 10-fold
higher potency than it inhibits other, related enzymes. Thus, the
NAP activity is selective toward that specified enzyme.
[0050] The term "substantially the same" when used to refer to
proteins, amino acid sequences, cDNAs, nucleotide sequences and the
like refers to proteins, cDNAs or sequences having at least about
90% homology with the other protein, cDNA, or sequence.
[0051] The term "NAP" or "NAP protein" means an isolated protein
which includes at least one NAP domain and having serine protease
inhibitory activity and/or anticoagulant activity.
BRIEF DESCRIPTION OF THE DRAWINGS
[0052] FIG. 1 depicts the nucleotide sequence of the AcaNAP5 cDNA
[SEQ. ID. NO. 3]. The numbering starts at the first nucleotide of
the cDNA. Translation starts at the first ATG codon (position 14);
a second in frame ATG is present at position 20.
[0053] FIG. 2 depicts the amino acid sequence of mature AcaNAP5
[SEQ. ID. NO. 4].
[0054] FIG. 3 depicts the nucleotide sequence of the AcaNAP6 cDNA
[SEQ. ID. NO. 5]. The numbering starts at the first nucleotide of
the cDNA. Translation starts at the first ATG codon (position 14);
a second in frame ATG is present at position 20.
[0055] FIG. 4 depicts the amino acid sequence of mature AcaNAP6
[SEQ. ID. NO. 6]. Amino acids that differ from AcaNAP5 are
underlined. In addition to these amino acid substitutions, AcaNAP6
contains a two amino acid deletion (Pro-Pro) when compared to
AcaNAP5.
[0056] FIG. 5 depicts the amino acid sequence of Pro-AcaNAP5 [SEQ.
ID. NO. 7].
[0057] FIG. 6 depicts the amino acid sequence of Pro-AcaNAP6 [SEQ.
ID. NO. 8]: Amino acids that differ from Pro-AcaNAP5 are
underlined. In addition to these amino acid substitutions,
Pro-AcaNAP6 contains a two amino acid deletion (Pro-Pro) when
compared to Pro-AcaNAP5.
[0058] FIGS. 7A through 7F depict the nucleotide sequences of the
cDNAs and deduced amino acid sequences of certain NAP proteins
isolated from Ancylostoma ceylanicum, Ancylostoma duodenale, and
Heligmosomoides polygyrus. FIG. 7A depicts sequences for the
recombinant cDNA molecule, AceNAP4, isolated from Ancylostoma
ceylanicum [SEQ. ID. NO. 9]. FIG. 7B depicts sequences for the
recombinant cDNA molecule, AceNAP5, isolated from Ancylostoma
ceylanicum [SEQ. ID. NO. 10]. FIG. 7C depicts sequences for the
recombinant cDNA molecule, AceNAP7, isolated from Ancylostoma
ceylanicum [SEQ. ID. NO. 11]. FIG. 7D depicts sequences for the
recombinant cDNA molecule, AduNAP4, isolated from Ancylostoma
duodenale [SEQ. ID. NO. 12]. FIG. 7E depicts sequences for the
recombinant cDNA molecule, AduNAP7, isolated from Ancylostoma
duodenale [SEQ. ID. NO. 13]. FIG. 7F depicts sequences for the
recombinant cDNA molecule, HpoNAP5, isolated from Heligmosomoides
polygyrus [SEQ. ID. NO. 14]. The EcoRI site, corresponding to the
5'-end of the recombinant cDNA molecule, is indicated in all cases
(underlined). Numbering of each sequence starts at this EcoRI site.
AceNAP4 and AduNAP7, each encode a protein which has two NAP
domains; all other clones in this Figure code for a protein having
a single NAP domain. The AduNAP4 cDNA clone is not full-length,
i.e., the recombinant cDNA molecule lacks the 5'-terminal part of
the coding region based on comparison with other isoforms.
[0059] FIGS. 8A through 8C depict the nucleotide sequence of the
vectors, pDONG61 (FIG. 8A) [SEQ. ID. NO. 15], pDONG62 (FIG. 8B)
[SEQ. ID. NO. 16], and pDONG63 (FIG. 8C) [SEQ. ID. NO. 17]. The
HindIII-BamHI fragment which is shown is located between the
HindIII and BamHI sites of pUC119. The vectors allow the cloning of
cDNAs, as SfiI-NotI fragments, in the three different reading
frames downstream of the filamentous phage gene 6. All relevant
restriction sites are indicated. The AAA Lys-encoding triplet at
position 373-375 is the last codon of gene 6. The gene 6 encoded
protein is followed by a Gly-Gly-Gly-Ser-Gly-Gly [SEQ. ID. NO. 18]
linker sequence.
[0060] FIG. 9 depicts the nucleotide sequence of the recombinant
cDNA molecule, AcaNAPc2 cDNA [SEQ. ID. NO. 19]. The EcoRI site,
corresponding to the 5'-end of the cDNA, is indicated (underlined).
Numbering starts at this EcoRI site. The deduced amino acid
sequence is also shown; the translational reading frame was
determined by the gene 6 fusion partner. The AcaNAPc2 cDNA lacks a
portion of the 5'-terminal part of the coding region; the homology
with AcaNAP5 and AcaNAP6 predicts that the first seven amino acid
residues belong to the secretion signal.
[0061] FIGS. 10A and 10B depict the comparative effects of certain
NAP proteins on the prothrombin time (PT) measurement (FIG. 10A)
and the activated partial thromboplastin time (aPTT) (FIG. 10B) of
normal citrated human plasma. Solid circles, (.circle-solid.),
represent Pro-AcaNAP5; open triangles, (.DELTA.), represent AcaNAP5
(AcaNAP5.sup.a in Table 2); and open circles, (.largecircle.),
represent native AcaNAP5.
[0062] FIG. 11 depicts the alignment of the amino acid sequences
encoded by certain NAP cDNAs isolated from various nematodes.
AcaNAP5 [SEQ. ID. NO. 20], AcaNAP6 [SEQ. ID. NO. 21], and AcaNAPc2
[SEQ. ID. NO. 128] were isolated from Ancylostoma caninum. AceNAP5
[SEQ. ID. NO. 22], AceNAP7 [SEQ. ID. NO. 23], and AceNAP4
(AceNAP4d1 [SEQ. ID. NO. 24] and AceNAP4d2 [SEQ. ID. NO. 25] were
isolated from Ancylostoma ceylanicum. AduNAP4 [SEQ. ID. NO. 26] and
AduNAP7 (AduNAP7d1 [SEQ. ID. NO. 27] and AduNAP7d2 [SEQ. ID. NO.
28]) were isolated from Ancylostoma duodenale. HpoNAP5 [SEQ. ID.
NO. 29] was isolated from Heligmosomoides polygyrus. The amino acid
sequences shown in this figure are as given in FIGS. 1, 3, 7A
through 7F, and 9. The sequences of mature AcaNAP5 [SEQ. ID. NO. 4]
and AcaNAP6 [SEQ. ID. NO. 6] (see FIGS. 2 and 4) are characterized,
in part, by ten cysteine residues (numbered one through ten and
shown in bold). All of the amino acid sequences in this Figure
contain at least one NAP domain. The AceNAP4 cDNA consists of two
adjacent regions, named AceNAP4d1 [SEQ. ID. NO. 24] and AceNAP4d2
[SEQ. ID. NO. 25], which encode a first (d1) and second (d2)
NAP-domain; similarly, the AduNAP7 cDNA contains two adjacent
regions, AduNAP7d1 [SEQ. ID. NO. 27] and AduNAP7d2 [SEQ. ID. NO.
28], encoding a first (d1) and second (d2) NAP-domain. The
alignment of the amino acid sequences of all NAP-domains is guided
by the cysteines; dashes ( - - - ) were introduced at certain
positions to maintain the cysteine alignment and indicate the
absence of an amino acid at that position. The carboxy-terminal
residue of a cDNA encoded protein is followed by the word
"end".
[0063] FIGS. 12A and 12B depict a map of the P. pastoris pYAM7SP8
expression/secretion vector (FIG. 12A) and sequences included in
the vector (FIG. 12B) [SEQ. ID. NO. 30]. As depicted in FIG. 12A,
this plasmid contains the following elements inserted between the
methanol-induced AOX1 promoter (dark arrow in the 5'AOX
untranslated region) and the AOX1 transcription termination signal
(3'T): a synthetic DNA fragment encoding the acid phosphatase
secretion signal (S), a synthetic 19-amino acid pro sequence (P)
ending with a Lys-Arg processing site for the KEX2 protease and a
multicloning site. The HIS4 gene which serves as a selection marker
in GS115 transformation was modified by site directed mutagenesis
to eliminate the Stu1 recognition sequence (HIS4*). pBR322
sequences, including the Bla gene and origin (ori) for propagation
in E. coli are represented by a single line. FIG. 12B depicts the
following contiquous DNA sequences which are incorporated in
pYAM7SP8: the acid phosphatase (PH01) secretion signal sequence,
pro sequence and multicloning site (MCS) sequence. The ATG start
codon of the PHO1 secretion signal is underlined.
[0064] FIGS. 13A through 13H depict the nucleotide sequences of the
cDNAs and deduced amino acid sequences of certain NAP proteins
isolated from Ancylostoma caninum. FIG. 13A depicts sequences for
the recombinant cDNA molecule AcaNAP23 [SEQ. ID. NO. 31]. FIG. 13B
depicts sequences for the recombinant cDNA molecule AcaNAP24 [SEQ.
ID. NO. 32]. FIG. 13C depicts sequences for the recombinant cDNA
molecule AcaNAP25 [SEQ. ID. NO. 33]. FIG. 13D depicts sequences for
the recombinant cDNA molecules AcaNAP31, AcaNAP42, and AcaNAP46,
all of which are identical [SEQ. ID. NO. 34]. FIG. 13E depicts
sequences for the recombinant cDNA molecule AcaNAP44 [SEQ. ID. NO.
35]. FIG. 13F depicts sequences for the recombinant cDNA molecule
AcaNAP45 [SEQ. ID. NO. 36]. FIG. 13G depicts sequences for the
recombinant cDNA molecule AcaNAP47 [SEQ. ID. NO. 37]. FIG. 13H
depicts sequences for the recombinant cDNA molecule AcaNAP48 [SEQ.
ID. NO. 38]. The EcoRI site, corresponding to the 5'-end of the
recombinant cDNA molecule, is indicated in all cases (underlined).
Numbering of each sequence starts at this EcoRI site. AcaNAP45 and
AcaNAP47, each encode a protein which has two NAP domains; all
other clones in this Figure code for a protein having a single NAP
domain.
[0065] FIG. 14 depicts the nucleotide, and deduced amino acid,
sequence of the recombinant cDNA molecule NamNAP [SEQ. ID. NO.
39].
[0066] FIG. 15 presents the antithrombotic activity of AcaNAP5 and
Low Molecular Weight Heparin (LMWH; Enoxaparin.TM.) evaluated in
the FeCl.sub.3 model of arterial thrombosis. Activity data is
represented as the percent incidence of occlusive thrombus
formation in the carotid artery (circles). Thrombus formation began
150 minutes after subcutaneous (s.c.) administration of test agent.
Deep wound bleeding was quantified in a separate group of animals
that were treated in an identical manner but without addition of
FeCl.sub.3 (squares). Blood loss at a deep surgical wound in the
neck was quantified over a total of 210 minutes after subcutaneous
compound administration.
[0067] FIG. 16 presents the alignment of amino acid sequences
corresponding to mature NAPs isolated according to the procedures
disclosed herein: namely AcaNAP5 [SEQ. ID. NO. 40], AcaNAP6 [SEQ.
ID. NO. 41], AcaNAP48 [SEQ. ID. NO. 42], AcaNAP23 [SEQ. ID. NO.
43], AcaNAP24 [SEQ. ID. NO. 44], AcaNAP25 [SEQ. ID. NO. 45],
AcaNAP44 [SEQ. ID. NO. 46], AcaNAP31, 42, 46 [SEQ. ID. NO. 47],
AceNAP4d1 [SEQ. ID. NO. 48], AceNAP4d2 [SEQ. ID. NO. 49],
AcaNAP45d1 [SEQ. ID. NO. 50], AcaNAP47d1 [SEQ. ID. NO. 51],
AduNAP7d1 [SEQ. ID. NO. 52], AcaNAP45d2 [SEQ. ID. NO. 53],
AcaNAP47d2 [SEQ. ID. NO. 54], AduNAP4 [SEQ. ID. NO. 55], AduNAP7d2
[SEQ. ID. NO. 56], AceNAP5 [SEQ. ID. NO. 57], AceNAP7 [SEQ. ID. NO.
58], AcaNAPc2 [SEQ. ID. NO. 59], HpoNAP5 [SEQ. ID. NO. 60], and
NamNAP [SEQ. ID. NO. 61]. Each NAP domain comprises ten cysteine
residues, which are used to align the sequences, and amino acid
sequences between the cysteines. A1 through A10 represent the amino
acid sequences between the cysteine residues.
[0068] FIG. 17 depicts the amino acid sequence of mature AceNAP4
[SEQ. ID. NO. 62] having two NAP domains.
[0069] FIG. 18 depicts the amino acid sequence of mature AcaNAP45
[SEQ. ID. NO. 63] having two NAP domains.
[0070] FIG. 19 depicts the amino acid sequence of mature AcaNAP47
[SEQ. ID. NO. 64] having two NAP domains.
[0071] FIG. 20 depicts the amino acid sequence for mature AduNAP7
[SEQ. ID. NO. 65] having two NAP domains.
DETAILED DESCRIPTION OF THE INVENTION
[0072] This invention provides a family of proteins, collectively
referred to as Nematode-extracted Anticoagulant Proteins (NAPs).
These proteins are so designated because the first member
originally isolated was extracted from a nematode, the canine
hookworm, Ancyclostoma caninum. However, the designation NAP or NAP
domain should not be considered to limit the proteins of the
present invention by this or other natural source.
[0073] Individual NAP proteins are characterized by having at least
one NAP domain and by having serine protease inhibitory and/or
anticoagulant activity. Such anticoagulant activity may be assessed
by increases in clotting time in both the PT and aPTT assays
described herein, by the inhibition of factor Xa or factor VIIa/TF
activity, or by demonstration of activity in vivo. Preferably,
blood or plasma used in such assays derives from species known to
be infected by nematodes, such as pigs, humans, primates, and the
like. The NAP domain is an amino acid sequence. It is believed that
the NAP domain is responsible for the observed inhibitory and/or
anticoagulant activity. Certain representative NAP domains include
the amino acid sequences depicted in FIGS. 11 and 16, particularly
the sequences between the cysteines designated as Cysteine 1 and
Cysteine 10 in the Figures and the sequence following Cysteine 10.
The characteristics broadly defining this family of proteins, as
well as the nucleic acid molecules, including mRNAs sequences and
DNA sequences which encode such proteins, are provided. Methods of
making these proteins, as well as methods of making nucleic acid
molecules encoding such proteins, are also provided. The specific
examples provided are exemplary only and other members of the NAP
family of proteins, as well as nucleic acid sequences encoding
them, can be obtained by following the procedures outlined in these
examples and described herein.
[0074] The proteins of the present invention include isolated NAPs
which comprise proteins having anticoagulant activity and including
at least one NAP domain.
[0075] With respect to "anticoagulant activity", the purified
proteins of the present invention are active as anticoagulants, and
as such, are characterized by inhibiting the clotting of blood
which includes the clotting of plasma. In one aspect, the preferred
isolated proteins of the present invention include those which
increase the clotting time of human plasma as measured in both the
prothrombin time (PT) and activated partial thromboplastin time
(aPTT) assays.
[0076] In the PT assay, clotting is initiated by the addition of a
fixed amount of tissue factor-phospholipid micelle complex
(thromboplastin) to human plasma. Anticoagulants interfere with
certain interactions on the surface of this complex and increase
the time required to achieve clotting relative to the clotting
observed in the absence of the anticoagulant. The measurement of PT
is particularly relevant for assessing NAP anticoagulant activity
because the series of specific biochemical events required to cause
clotting in this assay are similar to those that must be overcome
by the hookworm in nature to facilitate feeding. Thus, the ability
of NAP to act as an inhibitor in this assay can parallel its
activity in nature, and is predictive of anticoagulant activity in
vivo. In both the assay and in nature, the coagulation response is
initiated by the formation of a binary complex of the serine
protease factor VIIa (fVIIa) and the protein tissue factor (TF)
(fVIIa/TF), resulting in the generation of fXa. The subsequent
assembly of fXa into the prothrombinase complex is the key event
responsible for the formation of thrombin and eventual clot
formation.
[0077] In the aPTT assay, clotting is initiated by the addition of
a certain fixed amount of negatively charged phospholipid micelle
(activator) to the human plasma. Substances acting as
anticoagulants will interfere with certain interactions on the
surface of the complex and again increase the time to achieve a
certain amount of clotting relative to that observed in the absence
of the anticoagulant. Example B describes such PT and aPTT assays.
These assays can be used to assess anticoagulant activity of the
isolated NAPs of the present invention.
[0078] The preferred isolated NAPs of the present invention include
those which double the clotting time of human plasma in the PT
assay when present at a concentration of about 1 to about 500
nanomolar and which also double the clotting time of human plasma
in the aPTT assay when present at a concentration of about 1 to
about 500 nanomolar. Especially preferred are those proteins which
double the clotting time of human plasma in the PT assay when
present at a concentration of about 5 to about 100 nanomolar, and
which also double the clotting time of human plasma in the aPTT
assay when present at a concentration of about 5 to about 200
nanomolar. More especially preferred are those proteins which
double the clotting time of human plasma in the PT assay when
present at a concentration about 10 to about 50 nanomolar, and
which also double the clotting time of human plasma in the aPTT
assay when present at a concentration of about 10 to about 100
nanomolar.
[0079] Anticoagulant, or antithrombotic, activity of NAPs of the
present invention also can be evaluated using the in vivo models
presented in Example F. The rat FeCl.sub.3 model described in part
A of that Example is a model of platelet dependent, arterial
thrombosis that is commonly used to assess antithrombotic
compounds. The model evaluates the ability of a test compound to
prevent the formation of an occlusive thrombus induced by
FeCl.sub.3 in a segment of the rat carotid artery. NAPs of the
present invention are effective anticoagulants in this model when
administered intravenously or subcutaneously. The deep wound
bleeding assay described in part B of Example F allows measurement
of blood loss after administration of an anticoagulant compound. A
desired effect of an anticoagulant is that it inhibits blood
coagulation, or thrombus formation, but not so much as to prevent
clotting altogether and thereby potentiate bleeding. Thus, the deep
wound bleeding assay measures the amount of blood loss over the 3.5
hour period after administration of anticoagulant. The data
presented in FIG. 15 show NAP of the present invention to be an
effective antithrombotic compound at a dose that does not cause
excessive bleeding. In contrast, the dose of low molecular weight
heparin (LMWH) that correlated with 0% occlusion caused about three
times more bleeding than the effective dose of NAP.
[0080] General NAP Domain [FORMULA I]
[0081] With respect to "NAP domain", the isolated proteins (or
NAPs) of the present invention include at least one NAP domain in
their amino acid sequence. Certain NAP domains have an amino acid
sequence having a molecular weight of about 5.0 to 10.0
kilodaltons, preferably from about 7.0 to 10.0 kilodaltons, and
containing 10 cysteine amino acid residues.
[0082] Certain preferred isolated NAPs of the present invention
include those which contain at least one NAP domain, wherein each
such NAP domain is further characterized by including the amino
acid sequence:
Cys-A.sub.1-Cys-A.sub.2-Cys-A.sub.3-Cys-A.sub.4-Cys-A.sub.5-Cys-A.sub.6-C-
ys-A.sub.7-Cys-A.sub.8-Cys-A.sub.9-Cys ("FORMULA I"),
[0083] wherein: (a) A.sub.1 is an amino acid sequence containing 7
to 8 amino acid residues; (b) A.sub.2 is an amino acid sequence
containing 2 to 5 amino acid residues; (c) A3 is an amino acid
sequence containing 3 amino acid residues; (d) A.sub.4 is an amino
acid sequence containing 6 to 17 amino acid residues; (e) A.sub.5
is an amino acid sequence containing 3 to 4 amino acid residues;
(f) A.sub.6 is an amino acid sequence containing 3 to 5 amino acid
residues; (g) A.sub.7 is an amino acid residue; (h) A.sub.8 is an
amino acid sequence containing 10 to 12 amino acid residues; and
(i) A.sub.9 is an amino acid sequence containing 5 to 6 amino acid
residues. Other NAPs having slightly different NAP domains (See
FORMULAS II to V) are encompassed within the present invention.
[0084] Especially preferred NAP domains include those wherein
A.sub.2 is an amino acid sequence containing 4 to 5 amino acid
residues and A.sub.4 is an amino acid sequence containing 6 to 16
amino acid residues. More preferred are NAP domains wherein: (a)
A.sub.1 has Glu as its fourth amino acid residue; (b) A.sub.2 has
Gly as its first amino acid residue; (c) A.sub.8 has Gly as its
third amino acid residue and Arg as its sixth amino acid residue;
and (d) A.sub.9 has Val as its first amino acid residue. More
preferably, A.sub.3 has Asp or Glu as its first amino acid residue
and Lys or Arg as its third amino acid residue and A.sub.7 is Val
or Gln. Also, more preferably A.sub.8 has Leu or Phe as its fourth
amino acid residue and Lys or Tyr as its fifth amino acid residue.
Also preferred are NAP domains where, when A.sub.8 has 11 or 12
amino acid residues, Asp or Gly is its penultimate amino acid
residue, and, where when A.sub.8 has 10 amino acids, Gly is its
tenth amino acid residue. For expression of recombinant protein in
certain expression systems, a recombinant NAP may additionally
include an amino acid sequence for an appropriate secretion signal.
Certain representative NAP domains include the sequences depicted
in FIG. 11 and FIG. 16, particularly the sequences between (and
including) the cysteines designated as Cysteine 1 and Cysteine 10
and following Cysteine 10.
[0085] According to a preferred aspect, provided are NAPs which
include at least one NAP domain of Formula I wherein the NAP domain
includes the amino acid sequence:
Cys-A1-Cys-A2-Cys-A3-Cys-A4-Cys-A5-Cys-A6-Cys-A7-Cys-
-A8-Cys-A9-Cys-A10 wherein (a) Cys-A1 is selected from SEQ. ID.
NOS. 66 and 129; (b) Cys-A2-Cys is selected from one of SEQ. ID.
NOS. 130 to 133; (c) A3-Cys-A4 is selected from one of SEQ. ID.
NOS. 134 to 145; (d) Cys-A5 is selected from SEQ. ID. NOS. 146 and
147; (e) Cys-A6 is selected from one of SEQ. ID. NOS. 148 to 150;
(f) Cys-A7-Cys-A8 is selected from one of SEQ. ID. NOS. 151 to 153;
and (g) Cys-A9-Cys is selected from SEQ. ID. NOS. 154 and 155. Also
preferred are such proteins wherein Cys-A2-Cys is selected from
SEQ. ID. NOS. 130 and 131 and A3-Cys-A4 is selected from one of
SEQ. ID. NOS. 135 to 145. More preferred are those proteins having
NAP domains wherein SEQ. ID. NOS. 66 and 129 have Glu at location
5; SEQ. ID. NOS. 130 and 131 have Gly at location 2; SEQ. ID. NOS.
151 to 153 have Gly at location 6 and Arg at location 9; and SEQ.
ID. NOS. 154 and 155 have Val at location 2. More preferably SEQ.
ID. NOS. 151 to 153 have Val or Glu at location 2, Leu or Phe at
location 7 and/or Lys or Tyr at location 8. It is further preferred
that SEQ. ID. NO. 151 has Asp or Gly at location 14; SEQ. ID. NO.
152 has Asp or Gly at location 13; and SEQ. ID. NO. 153 has Gly at
location 13.
[0086] Certain NAPs of the present invention demonstrate
specificity toward inhibiting a particular component in the
coagulation cascade, such as fXa or the fVIIa/TF complex. The
specificity of a NAP's inhibitory activity toward a component in
the coagualtion cascade can be evaluated using the protocol in
Example D. There, the ability of a NAP to inhibit the activity of a
variety of serine proteases involved in coagulation is measured and
compared. The ability of a NAP to inhibit the fVIIa/TF complex also
can be assessed using the protocols in Example E, which measure the
ability of a NAP to bind fXa in either an inhibitory or
noninhibitory manner and to inhibit FVIIa when complexed with TF.
AcaNAP5 and AcaNAP6 are examples of proteins having NAP domains
that specifically inhibit fXa. AcaNAPc2 is a protein having a NAP
domain that demonstrates selective inhibition of the fVIIa/TF
complex when fXa, or a catalytically active or inactive derivative
thereof, is present.
[0087] NAPs having Anticoagulant Activity, Including NAPs having
Factor Xa Inhibitory Activity (FORMULA II)
[0088] Thus, in one aspect NAPs of the present invention also
include an isolated protein having anticoagulant activity,
including an isolated protein having Factor Xa inhibitory activity,
and having one or more NAP domains, wherein each NAP domain
includes the sequence:
Cys-A1-Cys-A2-Cys-A3-Cys-A4-Cys-A5-Cys-A6-Cys-A7-Cys-A8-Cys-A9-Cys-A10
("FORMULA II"),
[0089] wherein
[0090] (a) A1 is an amino acid sequence of 7 to 8 amino acid
residues;
[0091] (b) A2 is an amino acid sequence;
[0092] (c) A3 is an amino acid sequence of 3 amino acid
residues;
[0093] (d) A4 is an amino acid sequence;
[0094] (e) A5 is an amino acid sequence of 3 to 4 amino acid
residues;
[0095] (f) A6 is an amino acid sequence;
[0096] (g) A7 is an amino acid;
[0097] (h) A8 is an amino acid sequence of 11 to 12 amino acid
residues;
[0098] (i) A9 is an amino acid sequence of 5 to 7 amino acid
residues; and
[0099] (j) A10 is an amino acid sequence;
[0100] wherein each of A2, A4, A6 and A10 has an independently
selected number of independently selected amino acid residues and
each sequence is selected such that each NAP domain has in total
less than about 120 amino acid residues.
[0101] Pharmaceutical compositions comprising NAP proteins
according to this aspect, and methods of inhibiting blood
coagulation comprising administering NAP proteins according to this
aspect also are contemplated by this invention.
[0102] NAP proteins within this aspect of the invention have at
least one NAP domain. Preferred are NAPs having one or two NAP
domains. NAP proteins AcaNAP5 [SEQ. ID. NOS. 4 and 40] and AcaNAP6
[SEQ. ID. NOS. 6 and 41] have one NAP domain and are preferred NAPs
according to this aspect of the invention.
[0103] Preferred NAP proteins according to one embodiment of this
aspect of the invention are those in which A2 is an amino acid
sequence of 3 to 5 amino acid residues, A4 is an amino acid
sequence of 6 to 19 amino acid residues, A6 is an amino acid
sequence of 3 to 5 amino acid residues, and A10 is an amino acid
sequence of 5 to 25 amino acid residues.
[0104] Thus, according to one preferred aspect, provided are
isolated proteins having anticoagulant activity, including isolated
proteins having activity as Factor Xa inhibitors, having at least
one NAP domain of formula II which includes the following sequence:
Cys-A1-Cys-A2-Cys-A3-Cys-A4-Cys-A5-Cys-A6-Cys-A7-Cys-A8-Cys-A9-Cys-A10
wherein (a) Cys-A1 is selected from SEQ. ID. NOS. 67 and 156; (b)
Cys-A2-Cys is selected from one of SEQ. ID. NOS. 157 to 159; (c)
A3-Cys-A4 is selected from one of SEQ. ID. NOS. 160 to 173; (d)
Cys-A5 is selected from SEQ. ID. NOS. 174 and 175; (e) Cys-A6 is
selected from one of SEQ. ID. NOS. 176 to 178; (f) Cys-A7-Cys-A8 is
selected from SEQ. ID. NOS. 179 and 180; (g) Cys-A9 is selected
from one of SEQ. ID. NOS. 181 to 183; and (h) Cys-A10 is selected
from one of SEQ. ID. NOS. 184. to 204.
[0105] In another preferred embodiment of this aspect of the
invention, A3 has the sequence Glu-A3.sub.a-A3.sub.b, wherein
A3.sub.a and A3.sub.b are independently selected amino acid
residues. More preferably, A3.sub.a is selected from the group
consisting of Ala, Arg, Pro, Lys, Ile, His, Leu, and Thr, and
A3.sub.b is selected from the group consisting of Lys, Thr, and
Arg. Especially preferred A3 sequences are selected from the group
consisting of Glu-Ala-Lys, Glu-Arg-Lys, Glu-Pro-Lys, Glu-Lys-Lys,
Glu-Ile-Thr, Glu-His-Arg, Glu-Leu-Lys, and Glu-Thr-Lys.
[0106] In an additional preferred embodiment of this aspect of the
invention, A4 is an amino acid sequence having a net anionic
charge.
[0107] According to this aspect of the invention, a preferred A7
amino acid residue is Val or Ile.
[0108] Another preferred embodiment of this aspect of the invention
is one in which A8 includes the amino acid sequence
A8.sub.a-A8.sub.b-A8.sub.c-A- 8.sub.d-A8.sub.e-A8.sub.f-A8.sub.g
[SEQ. ID. NO. 68], wherein
[0109] (a) A.sup.8a is the first amino acid residue in A8,
[0110] (b) at least one of A8.sub.a and A8.sub.b is selected from
the group consisting of Glu or Asp, and
[0111] (c) A8.sub.c through A8.sub.g are independently selected
amino acid residues.
[0112] Preferably, A8.sub.c is Gly, A8.sub.d is selected from the
group consisting of Phe, Tyr, and Leu, A8.sub.e is Tyr, A8.sub.f is
Arg, and A8.sub.g is selected from Asp and Asn. An especially
preferred A8.sub.c-A8.sub.d-A8.sub.e-A8.sub.f-A8.sub.g sequence is
selected from the group consisting of Gly-Phe-Tyr-Arg-Asp [SEQ. ID.
NO. 69], Gly-Phe-Tyr-Arg-Asn [SEQ. ID. NO. 70], Gly-Tyr-Tyr-Arg-Asp
[SEQ, ID. NO. 71], Gly-Tyr-Tyr-Arg-Asn [SEQ. ID. NO. 72], and
Gly-Leu-Tyr-Arg-Asp [SEQ. ID. NO. 73].
[0113] An additional preferred embodiment is one in which A10
includes an amino sequence selected from the group consisting of
Glu-Ile-Ile-His-Val [SEQ. ID. NO. 74], Asp-Ile-Ile-Met-Val [SEQ.
ID. NO. 75], Phe-Ile-Thr-Phe-Ala-Pro [SEQ. ID. NO. 76], and
Met-Glu-Ile-Ile-Thr [SEQ. ID. NO. 77].
[0114] NAP proteins AcaNAP5 and AcaNAP6 include the amino acid
sequence Glu-Ile-Ile-His-Val [SEQ. ID. NO. 74] in A10, and are
preferred NAPs according to this embodiment of the invention.
[0115] In one embodiment of this aspect of the invention, a
preferred NAP molecule is one wherein
[0116] (a) A3 has the sequence Glu-A3.sup.a-A3.sup.b, wherein
A3.sup.a and A3.sup.b are independently selected amino acid
residues;
[0117] (b) A4 is an amino acid sequence having a net anionic
charge;
[0118] (c) A7 is selected from the group consisting of Val and
Ile;
[0119] (d) A8 includes an amino acid sequence selected from the
group consisting of Gly-Phe-Tyr-Arg-Asp [SEQ. ID. NO. 69],
Gly-Phe-Tyr-Arg-Asn [SEQ. ID. NO. 70], Gly-Tyr-Tyr-Arg-Asp [SEQ.
ID. NO. 71], Gly-Tyr-Tyr-Arg-Asn [SEQ. ID. NO. 72], and
Gly-Leu-Tyr-Arg-Asp [SEQ. ID. NO. 73]; and
[0120] (e) A10 includes an amino sequence selected from the group
consisting of Glu-Ile-Ile-His-Val [SEQ. ID. NO. 74],
Asp-Ile-Ile-Met-Val [SEQ. ID. NO. 75], Phe-Ile-Thr-Phe-Ala-Pro
[SEQ. ID. NO. 76], and Met-Glu-Ile-Ile-Thr [SEQ. ID. NO. 77].
[0121] Pharmaceutical compositions comprising NAP proteins
according to this embodiment, and methods of inhibiting blood
coagulation comprising administering NAP proteins according to this
embodiment also are contemplated by this invention. NAP proteins
within this embodiment of the invention have at least one NAP
domain. Preferred are NAPs having one or two NAP domains. NAP
proteins AcaNAP5 and AcaNAP6 have one NAP domain and are preferred
NAPs according to this embodiment of the invention.
[0122] In another preferred embodiment, a NAP molecule is one
wherein
[0123] (a) A3 is selected from the group consisting of Glu-Ala-Lys,
Glu-Arg-Lys, Glu-Pro-Lys, Glu-Lys-Lys, Glu-Ile-Thr, Glu-His-Arg,
Glu-Leu-Lys, and Glu-Thr-Lys;
[0124] (b) A4 is an amino acid sequence having a net anionic
charge;
[0125] (c) A7 is Val or Ile;
[0126] (d) A8 includes an amino acid sequence selected from the
group consisting of A8.sub.a-A8.sub.b-Gly-Phe-Tyr-Arg-Asp [SEQ. ID.
NO. 78], A8.sub.a-A8.sub.b-Gly-Phe-Tyr-Arg-Asn [SEQ. ID. NO. 79],
A8.sub.a-A8.sub.b-Gly-Tyr-Tyr-Arg-Asp [SEQ. ID. NO. 80],
A.sup.8.sub.a-A8.sub.b-Gly-Tyr-Tyr-Arg-Asn [SEQ. ID. NO. 81], and
A8.sub.a-A8.sub.b-Gly-Leu-Tyr-Arg-Asp [SEQ. ID. NO. 82], wherein at
least one of A.sup.8.sub.a and A8.sub.b is Glu or Asp;
[0127] (e) A9 is an amino acid sequence of five amino acid
residues; and
[0128] (f) A10 includes an amino acid sequence selected from the
group consisting of Glu-Ile-Ile-His-Val [SEQ. ID. NO. 74],
Asp-Ile-Ile-Met-Val [SEQ. ID. NO. 75], Phe-Ile-Thr-Phe-Ala-Pro
[SEQ. ID. NO. 76], and Met-Glu-Ile-Ile-Thr [SEQ. ID. NO. 77].
Pharmaceutical compositions comprising NAP proteins according to
this embodiment, and methods of inhibiting blood coagulation
comprising administering NAP proteins according to this embodiment
also are contemplated by this invention. NAP proteins within this
embodiment of the invention have at least one NAP domain. Preferred
are NAPs having one or two NAP domains. Preferred are proteins
having at least one NAP domain that is substantially the same as
that of either AcaNAP5 [SEQ. ID. NO. 40] or AcaNAP6 [SEQ. ID. NO.
41]. NAP proteins AcaNAP5 [SEQ. ID. NOS. 4 and 40] and AcaNAP6
[SEQ. ID. NOS. 6 and 41] have one NAP domain and are especially
preferred NAPs according to this embodiment of the invention.
[0129] Preferred NAP proteins having anticoagulant activity,
including those having Factor Xa inhibitory activity, according to
all the embodiments recited above for this aspect of the invention,
can be derived from a nematode species. A preferred nematode
species is selected from the group consisting of Ancylostoma
caninum, Ancylostoma ceylanicum, Ancylostoma duodenale, Necator
americanus, and Heligomosomoides polygyrus. Particularly preferred
are NAP proteins AcaNAP5 and AcaNAP6 derived from Ancylostoma
caninum.
[0130] This aspect of the invention also contemplates isolated
recombinant cDNA molecules encoding a protein having anticoagulant
and/or Factor Xa inhibitory activity, wherein the protein is
defined according to each of the embodiments recited above for
isolated NAP protein having anticoagulant and/or Factor Xa
inhibitory activity. Preferred cDNAs according to this aspect of
the invention code for AcaNAP5 and AcaNAP6.
[0131] The Factor Xa inhibitory activity of NAPs within this aspect
of the invention can be determined using protocols described
herein. Example A describes one such method. In brief, a NAP is
incubated with factor Xa for a period of time, after which a factor
Xa substrate is added. The rate of substrate hydrolysis is
measured, with a slower rate compared to the rate in the absence of
NAP indicative of NAP inhibition of factor Xa. Example C provides
another method of detecting a NAP's inhibitory activity toward
factor Xa when it is assembled into the prothrombinase complex,
which more accurately reflects the normal physiological function of
fXa in vivo. As described therein, factor Xa assembled in the
prothrombinase complex is incubated with NAP, followed by addition
of substrate. Factor Xa-mediated thrombin generation by the
prothrombinase complex is measured by the rate of thrombin
generation from this mixture.
[0132] NAPs having Anticoagulant Activity, Including NAPs having
Factor VIIa/TF Inhibitory Activity (FORMULA III)
[0133] In another aspect, NAPs of the present invention also
include an isolated protein having anticoagulant activity,
including and isolated protein having Factor VIIa/TF inhibitory
activity and having one or more NAP domains, wherein each NAP
domain includes the sequence:
Cys-A1-Cys-A2-Cys-A3-Cys-A4-Cys-A5-Cys-A6-Cys-A7-Cys-A8-Cys-A9-Cys-A10
("FORMULA III"),
[0134] wherein
[0135] (a) A1 is an amino acid sequence of 7 to 8 amino acid
residues;
[0136] (b) A2 is an amino acid sequence;
[0137] (c) A3 is an amino acid sequence of 3 amino acid
residues;
[0138] (d) A4 is an amino acid sequence;
[0139] (e) A5 is an amino acid sequence of 3 to 4 amino acid
residues;
[0140] (f) A6. is an amino acid sequence;
[0141] (g) A7 is an amino acid;
[0142] (h) A8 is an amino acid sequence of 11 to 12 amino acid
residues;
[0143] (i) A9 is an amino acid sequence of 5 to 7 amino acid
residues; and
[0144] (j) A10 is an amino acid sequence;
[0145] wherein each of A2, A4, A6 and A10 has an independently
selected number of independently selected amino acid residues and
each sequence is selected such that each NAP domain has in total
less than about 120 amino acid residues.
[0146] Pharmaceutical compositions comprising NAP proteins
according to this aspeact, and methods of inhibiting blood
coagulation comprising administering NAP proteins according to this
aspect also are contemplated by this invention. NAP proteins within
this aspect of the invention have at least one NAP domain.
Preferred are NAPs having one or two NAP domains. Preferred are
proteins having at least one NAP domain substantially the same as
that of AcaNAPc2 [SEQ. ID. NO. 59]. NAP protein AcaNAPc2 [SEQ. ID.
NO. 59] has one NAP domain and is an especially preferred NAP
according to this aspect of the invention.
[0147] Preferred NAP proteins according to this aspect of the
invention are those in which A2 is an amino acid sequence of 3 to 5
amino acid residues, A4 is an amino acid sequence of 6 to 19 amino
acid residues, A6 is an amino acid sequence of 3 to 5 amino acid
residues, and A10 is an amino acid sequence of 5 to 25 amino acid
residues.
[0148] Accordingly, in one preferred aspect, provided are NAPs
having anticoagulant activity, including factor VIIa/TF inhibitory
activity, and having at least one NAP domain of formula III wherein
the NAP domain includes the amino acid sequence:
Cys-A1-Cys-A2-Cys-A3-Cys-A4-Cys-A5-Cys--
A6-Cys-A7-Cys-A8-Cys-A9-Cys-A10 wherein (a) Cys-A1 is selected from
SEQ. ID. NOS. 83 and 205; (b) Cys-A2-Cys is selected from one of
SEQ. ID. NOS. 206 to 208; (c) A3-Cys-A4 is selected from one of
SEQ. ID. NOS. 209 to 222; (d) Cys-A5 is selected from SEQ. ID. NOS.
223 and 224; (e) Cys-A6 is selected from one of SEQ. ID. NOS. 225
to 227; (f) Cys-A7-Cys-A8 is selected from SEQ. ID. NOS. 228 and
229; (g) Cys-A9 is selected from SEQ. ID. NOS. 230 to 232; and (h)
Cys-A10 is selected from one of SEQ. ID. NOS. 233 to 253.
[0149] In another preferred embodiment according to this aspect of
the invention, A3 has the sequence Asp-A3.sub.a-A3.sub.b, wherein
A3.sub.a and A3.sub.b are independently selected amino acid
residues. More preferably, A3 is Asp-Lys-Lys.
[0150] In an additional preferred embodiment, A4 is an amino acid
sequence having a net anionic charge.
[0151] In another preferred embodiment of this aspect of the
invention, A5 has the sequence A5.sub.a-A5.sub.b-A5.sub.c-A5.sub.d
[SEQ. ID. NO. 84], wherein A5.sub.a through A5.sub.d are
independently selected amino acid residues. Preferably, A5.sub.a is
Leu and A5.sub.c is Arg.
[0152] According to this aspect of the invention, a preferred A7
amino acid residue is Val or Ile, more preferably Val.
[0153] An additional preferred embodiment of this aspect of the
invention is one in which A8 includes the amino acid sequence
A8.sub.a-A8.sub.b-A8.sub.c-A8.sub.d-A8.sub.e-A8.sub.f-A8.sub.g
[SEQ. ID. NO. 68], wherein
[0154] (a) A8.sub.a is the first amino acid residue in A8,
[0155] (b) at least one of A8a and A8b is selected from the group
consisting of Glu or Asp, and
[0156] (c) A8.sub.c through A8.sub.g are independently selected
amino acid residues.
[0157] Preferably, A8.sub.c is Gly, A8.sub.d is selected from the
group consisting of Phe, Tyr, and Leu, A8.sub.e is Tyr, A8.sub.f is
Arg, and A8.sub.g is selected from Asp and Asn. A preferred
A8.sub.c-A8.sub.d-A8.sub.e-A8.sub.f-A8.sub.g sequence is
Gly-Phe-Tyr-Arg-Asn [SEQ. ID. NO. 70].
[0158] In one embodiment, a preferred NAP molecule is one
wherein:
[0159] (a) A3 has the sequence Asp-A3.sub.a-A3.sub.b, wherein
A.sup.3.sub.a and A3.sub.b are independently selected amino acid
residues;
[0160] (b) A4 is an amino acid sequence having a net anionic
charge;
[0161] (c) A5 has the sequence A5.sub.a-A5.sub.b-A5.sub.c-A5.sub.d,
wherein A.sup.5.sub.a through A5.sub.d are independently selected
amino acid residues; and
[0162] (d) A7 is selected from the group consisting of Val and Ile.
Pharmaceutical compositions comprising NAP proteins according to
this embodiment, and methods of inhibiting blood coagulation
comprising administering NAP proteins according to this embodiment
also are contemplated by this invention. NAP proteins within this
embodiment of the invention have at least one NAP domain. Preferred
are NAPs having one or two NAP domains. NAP protein AcaNAPc2 has
one NAP domain and is a preferred NAP according to this embodiment
of the invention.
[0163] In another preferred embodiment, a NAP molecule is one
wherein
[0164] (a) A3 is Asp-Lys-Lys;
[0165] (b) A4 is an amino acid sequence having a net anionic
charge;
[0166] (c) AS has the sequence A5.sub.a-A5.sub.b-A5.sub.c-A5.sub.d
[SEQ. ID. NO. 85], wherein A5.sub.a through A5.sub.d are
independently selected amino acid residues;
[0167] (d) A7 is Val; and
[0168] (e) A8 includes an amino acid sequence
A8.sub.a-A8.sub.b-Gly-Phe-Ty- r-Arg-Asn [SEQ. ID. NO. 79], wherein
at least one of A.sup.8.sub.a and A8.sub.b is Glu or Asp.
Pharmaceutical compositions comprising NAP proteins according to
this embodiment, and methods of inhibiting blood coagulation
comprising administering NAP proteins according to this embodiment
also are contemplated by this invention. NAP proteins within this
embodiment of the invention have at least one NAP domain. Preferred
are NAPs having one or two NAP domains. NAP protein AcaNAPc2 [SEQ.
ID. NO. 59] has one NAP domain and is a preferred NAP according to
this embodiment of the invention.
[0169] Preferred NAP proteins having anticoagulant activity,
including those having Factor VIIa/TF inhibitory activity,
according to all the embodiments recited above for this aspect of
the invention, can be derived from a nematode species. A preferred
nematode species is selected from the group consisting of
Ancylostoma caninum, Ancylostoma ceylanicum, Ancylostoma duodenale,
Necator americanus, and Heligomosomoides polygyrus. Particularly
preferred is NAP protein AcaNAPc2 derived from Ancylostoma
caninum.
[0170] This aspect of the invention also contemplates isolated
recombinant cDNA molecules encoding a protein having anticoagulant
and/or Factor VIIa/TF inhibitory activity, wherein the protein is
defined according to each of the embodiments recited above for
isolated NAP protein having anticoagulant and/or Factor VIIa/TF
inhibitory activity. A preferred cDNA according to this aspect has
a nucleotide sequence [SEQ. ID. NO. 19] and codes for AcaNAPc2
[SEQ. ID. NO. 59].
[0171] The fVIIa/TF inhibitory activity of NAPs within this aspect
of the invention can be determined using protocols described
herein. Example E describes fVIIa/TF assays. There, the
fVIIa/TF-mediated cleavage and liberation of the tritiated
activation peptide from radiolabeled human factor IX (.sup.3H-FIX)
or the amidolytic hydrolysis of a chromogenic peptidyl substrate
are measured. Interestingly, NAP fVIIa/TF inhibitors of the present
invention require the presence of fXa in order to be active
fVIIa/TF inhibitors. However, NAP fVIIa/TF inhibitors were equally
effective in the presence of fXa in which the active site had been
irreversibly occupied with the peptidyl chloromethyl ketone
H-Glu-Gly-Arg-CMK (EGR), and thereby rendered catalytically
inactive (EGR-fXa). While not wishing to be bound by any one
explanation, it appears that a NAP having fVIIa/TF inhibition
activity forms a binary complex with fXa by binding to a specific
recognition site on the enzyme that is distinct from the primary
recognition sites P.sub.4-P.sub.1, within the catalytic center of
the enzyme. This is followed by the formation of a quaternary
inhibitory complex with the fVIIa/TF complex. Consistent with this
hypothesis is that EGR-fXa can fully support the inhibition of
fVIIa/TF by NAPs inhibitory for fVIIa/TF despite covalent occupancy
of the primary recognition sites (P.sub.4-P.sub.1) within the
catalytic site of fXa by the tripeptidyl-chloromethyl ketone
(EGR-CMK).
[0172] The fVIIa/TF inhibitory activity of NAPs also can be
determined using the protocols in Example D, as well as the fXa
assays described in Examples A and C. There, the ability of a NAP
to inhibit the catalytic activity of a variety of enzymes is
measured and compared to its inhibitory activity toward the
fVIIa/TF complex. Specific inhibition of fVIIa/TF by a NAP is a
desired characteristic for certain applications.
[0173] A further aspect of the invention includes an isolated
protein having anticoagulant activity, and cDNAs coding for the
protein, wherein said protein specifically inhibits the catalytic
activity of the fVIIa/TF complex in the presence of fXa or
catalytically inactive fXa derivative, but does not specifically
inhibit the activity of FVIIa in the absence of TF and does not
specifically inhibit prothrombinase. Preferred proteins according
to this aspect of the invention have the characteristics described
above for an isolated protein having Factor VIIa/TF inhibitory
activity and having one or more NAP domains. A preferred protein
according to this aspect of the invention is AcaNAPc2.
[0174] NAPs within this aspect of the invention are identified by
their fVIIa/TF inhibitory activity in the presence of fXa or a fXa
derivative, whether the derivative is catalytically active or not.
The protocols described in Examples B, C, and F are useful in
determining the anticoagulant activity of such NAPs. The protocol
in Example A can detect a NAP's inactivity toward free fXa or
prothrombinase. Data generated using the protcols in Example E will
identify NAPs that require either catalytically active or inactive
fXa to inhibit fVIIa/TF complex.
[0175] NAPs having Serine Protease Inhibitory Activity (FORMULA
IV)
[0176] In an additional aspect, NAPs of the present invention also
include an isolated protein having serine protease inhibitory
activity and having one or more NAP domains, wherein each NAP
domain includes the sequence:
Cys-A1-Cys-A2-Cys-A3-Cys-A4-Cys-A5-Cys-A6-Cys-A7-Cys-A8-Cys-A9-Cys-A10,
("FORMULA IV") wherein
[0177] (a) A1 is an amino acid sequence of 7 to 8 amino acid
residues;
[0178] (b) A2 is an amino acid sequence;
[0179] (c) A3 is an amino acid sequence of 3 amino acid
residues;
[0180] (d) A4 is an amino acid sequence;
[0181] (e) A5 is an amino acid sequence of 3 to 4 amino acid
residues;
[0182] (f) A6 is an amino acid sequence;
[0183] (g) A7 is an amino acid;
[0184] (h) A8 is an amino acid sequence of 10 to 12 amino acid
residues;
[0185] (i) A9 is an amino acid sequence of 5 to 7 amino acid
residues; and
[0186] (j) A10 is an amino acid sequence;
[0187] wherein each of A2, A4, A6 and A10 has an independently
selected number of independently selected amino acid residues and
each sequence is selected such that each NAP domain has in total
less than about 120 amino acid residues. Pharmaceutical
compositions comprising NAP proteins according to this aspect, and
methods of inhibiting blood coagulation comprising administering
NAP proteins according to this aspect also are contemplated by this
invention. NAP proteins within this aspect of the invention have at
least one NAP domain. Preferred are NAPs having one or two NAP
domains. Preferred are NAP domains that have amino acid sequences
that are substantially the same as the NAP domains of HpoNAP5 [SEQ.
ID. NO. 60] or NamNAP [SEQ. ID. NO. 61]. NAP proteins HpoNAP5 [SEQ.
ID. NO. 60] and NamNAP [SEQ. ID. NO. 61] have one NAP domain and
are preferred NAPs according to this aspect of the invention.
[0188] Preferred NAP proteins according to this aspect of the
invention are those in which A2 is an amino acid sequence of 3 to 5
amino acid residues, A4 is an amino acid sequence of 6 to 19 amino
acid residues, A6 is an amino acid sequence of 3 to 5 amino acid
residues, and A10 is an amino acid sequence of 1 to 25 amino acid
residues.
[0189] Thus, in one preferred aspect, NAPs exhibiting serine
protease activity have at least one NAP domain of Formula IV which
includes the amino acid sequence:
Cys-A1-Cys-A2-Cys-A3-Cys-A4-Cys-A5-Cys-A6-Cys-A7-Cys-
-A8-Cys-A9-Cys-A10 wherein (a) Cys-A1 is selected from SEQ. ID.
NOS. 86 and 254; (b) Cys-A2-Cys is selected from one of SEQ. ID.
NOS. 255 to 257; (c) A3-Cys-A4 is selected from one of SEQ. ID.
NOS. 258 to 271; (d) Cys-A5 is selected from SEQ. ID. NOS. 272 and
273; (e) Cys-A6 is selected from one of SEQ. ID. NOS. 274 to 276;
(f) Cys-A7-Cys-A8 is selected from one of SEQ. ID. NOS. 277 to 279;
(g) Cys-A9 is selected from one of SEQ. ID. NOS. 280 to 282; and
(h) Cys-A10 is selected from one of SEQ. ID. NOS. 283 to 307.
[0190] In another preferred embodiment, A3 has the sequence
Glu-A3.sub.a-A3.sub.b, wherein A3.sub.a and A3.sub.b are
independently selected amino acid residues. More preferably, A3 is
Glu-Pro-Lys.
[0191] In an additional preferred embodiment, A4 is an amino acid
sequence having a net anionic charge.
[0192] In another preferred embodiment, A5 has the sequence
A5.sub.a-A5.sub.b-A5.sub.c, wherein A5.sub.a through A5.sub.c are
independently selected amino acid residues. Preferably, A5.sub.a is
Thr and A5.sub.c is Asn. An especially preferred A5 sequence
includes Thr-Leu-Asn or Thr-Met-Asn.
[0193] According to this aspect of the invention, a preferred A7
amino acid residue is Gln.
[0194] In one embodiment of this aspect of the invention, a
preferred NAP molecule is one wherein
[0195] (a) A3 has the sequence Glu-A3.sub.a-A3.sub.b, wherein
A3.sub.a and A3.sub.b are independently selected amino acid
residues;
[0196] (b) A4 is an amino acid sequence having a net anionic
charge;
[0197] (c) A5 has the sequence A5.sub.a-A.sup.5.sub.b-A5.sub.c,
wherein A5.sub.a through A.sup.5.sub.c are independently selected
amino acid residues, and
[0198] (d) A7 is Gln. Pharmaceutical compositions comprising NAP
proteins according to this embodiment, and methods of inhibiting
blood coagulation comprising administering NAP proteins according
to this embodiment also are contemplated by this invention. NAP
proteins within this embodiment of the invention have at least one
NAP domain. Preferred are NAPs having one or two NAP domains. NAP
proteins HpoNAP5 [SEQ. ID. NO. 60] and NamNAP [SEQ. ID. NO. 61]
have one NAP domain and are preferred NAPs according to this
embodiment of the invention.
[0199] In another preferred embodiment, a NAP molecule is one
wherein
[0200] (a) A3 is Glu-Pro-Lys;
[0201] (b) A4 is an amino acid sequence having a net anionic
charge;
[0202] (c) A5 is selected from Thr-Leu-Asn and Thr-Met-Asn; and
[0203] (d) A7 is Gln. Pharmaceutical compositions comprising NAP
proteins according to this embodiment, and methods of inhibiting
blood coagulation comprising administering NAP proteins according
to this embodiment also are contemplated by this invention. NAP
proteins within this embodiment of the invention have at least one
NAP domain. Preferred are NAPs having one or two NAP domains. NAP
proteins HpoNAP5 [SEQ. ID. NO. 60] and NamNAP [SEQ. ID. NO. 61]
have one NAP domain and are preferred NAPs according to this
embodiment of the invention.
[0204] Preferred NAP proteins having serine protease inhibitory
activity, according to all the embodiments recited above for this
aspect of the invention, can be derived from a nematode species. A
preferred nematode species is selected from the group consisting of
Ancylostoma caninum, Ancylostoma ceylanicum, Ancylostoma duodenale,
Necator americanus, and Heligomosomoides polygyrus. Particularly
preferred are NAP proteins HpoNAP5 and NamNAP derived from
Heligomosomoides polygyrus and Necator americanus,
respectively.
[0205] This aspect of the invention also contemplates isolated
recombinant cDNA molecules encoding a protein having serine
protease inhibitory activity, wherein the protein is defined
according to each of the embodiments recited above for isolated NAP
protein having serine protease inhibitory activity. Preferred cDNAs
according to this aspect have nucleotide sequences [SEQ. ID. NO.
14] (HpoNAP5) and [SEQ. ID. NO. 39] (NamNAP) and code for HpoNAP5
[SEQ. ID. NO. 60] and NamNAP [SEQ. ID. NO. 61].
[0206] The serine protease inhibitory activity can be determined
using any of the assays disclosed in Examples A through F, or any
commonly used enzymatic assay for measuring inhibition of serine
protease activity. Procedures for a multitude of enzymatic assays
can be found in the volumes of Methods of Enzymology or similar
reference materials. Preferred NAPs have serine protease inhibitory
activity directed toward enzymes in the blood coagulation cascade
or toward trypsin/elastase.
[0207] NAPs having Anticoagulant Activity (FORMULA V)
[0208] In another aspect of the invention, NAPs of the present
invention also include an isolated protein having anticoagulant
activity and having one or more NAP domains, wherein each NAP
domain includes the sequence:
Cys-A1-Cys-A2-Cys-A3-Cys-A4-Cys-A5-Cys-A6-Cys-A7-Cys-A8-Cys-A9-Cys-A10
("FORMULA V"), wherein
[0209] (a) A1 is an amino acid sequence of 7 to 8 amino acid
residues;
[0210] (b) A2 is an amino acid sequence;
[0211] (c) A3 is an amino acid sequence of 3 amino acid
residues;
[0212] (d) A4 is an amino acid sequence;
[0213] (e) A5 is an amino acid sequence of 3 to 4 amino acid
residues;
[0214] (f) A6 is an amino acid sequence;
[0215] (g) A7 is an amino acid;
[0216] (h) A8 is an amino acid sequence of 11 to 12 amino acid
residues;
[0217] (i) A9 is an amino acid sequence of 5 to 7 amino acid
residues; AND
[0218] (j) A10 is an amino acid sequence;
[0219] wherein each of A2, A4, A6 and A10 has an independently
selected number of independently selected amino acid residues and
each sequence is selected such that each NAP domain has in total
less than about 120 amino acid residues. Pharmaceutical
compositions comprising NAP proteins according to this aspeact, and
methods of inhibiting blood coagulation comprising administering
NAP proteins according to this aspect also are contemplated by this
invention. NAP proteins within this aspect of the invention have at
least one NAP domain. Preferred are NAPs having one or two NAP
domains. Preferred NAPs include those having at least one NAP
domain having an amino acid sequence substantially the same as any
of [SEQ. ID. NOS. 40 to 58]. NAP proteins AcaNAP5 [SEQ. ID. NO.
40], AcaNAP6 [SEQ. ID. NO. 41], AcaNAP48 [SEQ. ID. NO. 42],
AcaNAP23 [SEQ. ID. NO. 43], AcaNAP24 [SEQ. ID. NO. 44], AcaNAP25
[SEQ. ID. NO. 45], AcaNAP44 [SEQ. ID. NO. 46], AcaNAP31 [SEQ. ID.
NO. 47], AduNAP4 [SEQ. ID. NO. 55], AceNAP5 [SEQ. ID. NO. 57], and
AceNAP7 [SEQ. ID. NO. 58] have one NAP domain and are preferred
NAPs according to this aspect of the invention. NAP proteins
AceNAP4 [SEQ. ID. NO. 62], AcaNAP45 [SEQ. ID. NO. 63], AcaNAP47
[SEQ. ID. NO. 64], and AduNAP7 [SEQ. ID. NO. 65] have two NAP
domains and are preferred NAPs according to this aspect of the
invention.
[0220] Preferred NAP proteins according to this aspect of the
invention are those in which A2 is an amino acid sequence of 3 to 5
amino acid residues, A4 is an amino acid sequence of 6 to 19 amino
acid residues, A6 is an amino acid sequence of 3 to 5 amino acid
residues, and A10 is an amino acid sequence of 5 to 25 amino acid
residues.
[0221] Preferred NAPs of the present invention according to this
aspect include isolated proteins having anticoagulant activity and
having at least one NAP domain of formula V which includes the
following sequence:
Cys-A1-Cys-A2-Cys-A3-Cys-A4-Cys-A5-Cys-A6-Cys-A7-Cys-A8-Cys-A9-Cys-A10
wherein (a) Cys-A1 is selected from SEQ. ID. NOS. 87 and 308; (b)
Cys-A2-Cys is selected from one of SEQ. ID. NOS. 309 to 311; (c)
A3-Cys-A4 is selected from one of SEQ. ID. NOS. 312 to 325; (d)
Cys-A5 is selected from SEQ. ID. NOS. 326 and 327; (e) Cys-A6 is
selected from one of SEQ. ID. NOS. 328 to 330; (f) Cys-A7-Cys-A8 is
selected from SEQ. ID. NOS. 331 to 332; (g) Cys-A9 is selected from
one of SEQ. ID. NOS. 333 to 335; and (h) Cys-A10 is selected from
one of SEQ. ID. NOS. 336 to 356.
[0222] In another preferred embodiment, A3 has the sequence
Glu-A3.sub.a-A3.sub.b, wherein A3.sub.a and A3.sub.b are
independently selected amino acid residues. More preferably,
A3.sub.a is selected from the group consisting of Ala, Arg, Pro,
Lys, Ile, His, Leu, and Thr, and A3.sub.b is selected from the
group consisting of Lys, Thr, and Arg. Especially preferred A3
sequences are selected from the group consisting of Glu-Ala-Lys,
Glu-Arg-Lys, Glu-Pro-Lys, Glu-Lys-Lys, Glu-Ile-Thr, Glu-His-Arg,
Glu-Leu-Lys, and Glu-Thr-Lys.
[0223] In an additional preferred embodiment, A4 is an amino acid
sequence having a net anionic charge.
[0224] According to this aspect of the invention, a preferred A7
amino acid residue is Val or Ile.
[0225] Another preferred embodiment of the invention is one in
which A8 includes the amino acid sequence
A8.sub.a-A8.sub.b-A8.sub.c-A8.sub.d-A.su-
p.8.sub.e-A8.sub.f-A8.sub.g [SEQ. ID. NO. 68], wherein
[0226] (a) A8.sub.a is the first amino acid residue in A8,
[0227] (b) at least one of A8.sub.a and A8.sub.b is selected from
the group consisting of Glu or Asp, and
[0228] (c) A8.sub.c through A8.sub.g are independently selected
amino acid residues.
[0229] Preferably, A8.sub.c is Gly, A8.sub.d is selected from the
group consisting of Phe, Tyr, and Leu, A8.sub.e is Tyr, A8.sub.f is
Arg, and A8.sub.g is selected from Asp and Asn. A preferred
A8.sub.c-A8.sub.d-A8.sub.e-A8.sub.f-A8.sub.g sequence is selected
from the group consisting of Gly-Phe-Tyr-Arg-Asp [SEQ. ID. NO. 69],
Gly-Phe-Tyr-Arg-Asn [SEQ. ID. NO. 70], Gly-Tyr-Tyr-Arg-Asp. [SEQ.
ID. NO. 71], Gly-Tyr-Tyr-Arg-Asn [SEQ. ID. NO. 72], and
Gly-Leu-Tyr-Arg-Asp [SEQ. ID. NO. 73].
[0230] Another preferred embodiment is one in which A10 includes an
amino sequence selected from the group consisting of
Glu-Ile-Ile-His-Val [SEQ. ID. NO. 74], Asp-Ile-Ile-Met-Val [SEQ.
ID. NO. 75], Phe-Ile-Thr-Phe-Ala-Pro [SEQ. ID. NO. 76], and
Met-Glu-Ile-Ile-Thr [SEQ. ID. NO. 77]. NAP proteins AcaNAP5 [SEQ.
ID. NOS. 4 and 40] and AcaNAP6 [SEQ. ID. NOS. 6 and 41] include the
amino acid sequence Glu-Ile-Ile-His-Val [SEQ. ID. NO. 74] in A10,
and are preferred NAPs according to this embodiment of the
invention. NAP protein AcaNAP48 [SEQ. ID. NO. 42] includes the
amino acid sequence Asp-Ile-Ile-Met-Val [SEQ. ID. NO. 75] in A10
and is a preferred NAP according to this embodiment of the
invention. NAP proteins AcaNAp23 [SEQ. ID. NO. 43], AcaNAP24 [SEQ.
ID. NO. 44], AcaNAP25 [SEQ. ID. NO. 45], AcaNAP44 [SEQ. ID. NO.
46], AcaNAP31 [SEQ. ID. NO. 47], and AceNAP4 [SEQ. ID. NO. 48, 49
AND 62] include the amino acid sequence Phe-Ile-Thr-Phe-Ala-Pro
[SEQ. ID. NO. 76] and are preferred NAPs according to this
embodiment of the invention. NAP proteins AcaNAP45 [SEQ. ID. NOS.
50, 53 AND 63], AcaNAP47 [SEQ. ID. NO. 51, 54 AND 64], AduNAP7
[SEQ. ID. NO. 52, 56 AND 65], AduNAP4 [SEQ. ID. NO. 55], AceNAP5
[SEQ. ID. NO. 57], and AceNAP7 [SEQ. ID. NO. 58] include the amino
acid sequence Met-Glu-Ile-Ile-Thr [SEQ. ID. NO. 77] and are
preferred NAPs according to this embodiment of the invention.
[0231] In one embodiment, a preferred NAP molecule is one
wherein
[0232] (a) A3 has the sequence Glu-A3.sub.a-A3.sub.b, wherein
A.sup.3.sub.a and A3.sub.b are independently selected amino acid
residues;
[0233] (b) A4 is an amino acid sequence having a net anionic
charge;
[0234] (c) A7 is selected from the group consisting of Val and
Ile;
[0235] (d) A8 includes an amino acid sequence selected from the
group consisting of Gly-Phe-Tyr-Arg-Asp [SEQ. ID. NO. 69],
Gly-Phe-Tyr-Arg-Asn [SEQ. ID. NO. 70], Gly-Tyr-Tyr-Arg-Asp [SEQ.
ID. NO. 71], Gly-Tyr-Tyr-Arg-Asn [SEQ. ID. NO. 72], and
Gly-Leu-Tyr-Arg-Asp [SEQ. ID. NO. 73]; and
[0236] (e) A10 includes an amino sequence selected from the group
consisting of Glu-Ile-Ile-His-Val [SEQ. ID. NO. 74],
Asp-Ile-Ile-Met-Val [SEQ. ID. NO. 75], Phe-Ile-Thr-Phe-Ala-Pro
[SEQ. ID. NO. 76], and Met-Glu-Ile-Ile-Thr [SEQ. ID. NO. 77].
Pharmaceutical compositions comprising NAP proteins according to
this embodiment, and methods of inhibiting blood coagulation
comprising administering NAP proteins according to this embodiment
also are contemplated by this invention. NAP proteins within this
aspect of the invention have at least one NAP domain. Preferred are
NAPs having one or two NAP domains. NAP proteins AcaNAP5 [SEQ. ID.
NOS. 4 and 40], AcaNAP6 [SEQ. ID. NOS. 6 and 41], AcaNAP48 [SEQ.
ID. NO. 42], AcaNAP23 [SEQ. ID. NO. 43], AcaNAP24 [SEQ. ID. NO.
44], AcaNAP25 [SEQ. ID. NO. 45], AcaNAP44 [SEQ. ID. NO. 46],
AcaNAP31 [SEQ. ID. NO. 47], AduNAP4 [SEQ. ID. NO. 55], AceNAP5
[SEQ. ID. NO. 57], and AceNAP7 [SEQ. ID. NO. 58] have one NAP
domain and are preferred NAPs according to this embodiment. NAP
proteins AceNAP4 [SEQ. ID. NO. 62], AcaNAP45 [SEQ. ID. NO. 63],
AcaNAP47 [SEQ. ID. NO. 64], and AduNAP7 [SEQ. ID. NO. 65] have two
NAP domains and are preferred NAPs according to this
embodiment.
[0237] In another preferred embodiment, a NAP molecule is one
wherein
[0238] (a) A3 is selected from the group consisting of Glu-Ala-Lys,
Glu-Arg-Lys, Glu-Pro-Lys, Glu-Lys-Lys, Glu-Ile-Thr, Glu-His-Arg,
Glu-Leu-Lys, and Glu-Thr-Lys;
[0239] (b) A4 is an amino acid sequence having a net anionic
charge;
[0240] (c) A7 is Val or Ile;
[0241] (d) A8 includes an amino acid sequence selected from the
group consisting of A8.sub.a-A8.sub.b-Gly-Phe-Tyr-Arg-Asp [SEQ. ID.
NO. 78], A8.sub.a-A8.sub.b-Gly-Phe-Tyr-Arg-Asn [SEQ. ID. NO. 79],
A8.sub.a-A8.sub.b-Gly-Tyr-Tyr-Arg-Asp [SEQ. ID. NO. 80],
A.sup.8.sub.a-A8.sub.b-Gly-Tyr-Tyr-Arg-Asn [SEQ. ID. NO. 81], and
A8.sub.a-A8.sub.b-Gly-Leu-Tyr-Arg-Asp [SEQ. ID. NO. 82], wherein at
least one of A8.sub.a and A8.sub.b is Glu or Asp;
[0242] (e) A9 is an amino acid sequence of five amino acid
residues; and
[0243] (f) A10 includes an amino acid sequence selected from the
group consisting of Glu-Ile-Ile-His-Val [SEQ. ID. NO. 74],
Asp-Ile-Ile-Met-Val [SEQ. ID. NO. 75], Phe-Ile-Thr-Phe-Ala-Pro
[SEQ. ID. NO. 76], and Met-Glu-Ile-Ile-Thr [SEQ. ID. NO. 77].
Pharmaceutical compositions comprising NAP proteins according to
this embodiment, and methods of inhibiting blood coagulation
comprising administering NAP proteins according to this embodiment
also are contemplated by this invention. NAP proteins within this
embodiment of the invention have at least one NAP domain. Preferred
are NAPs having one or two NAP domains. NAP proteins AcaNAP5 [SEQ.
ID. NOS. 4 and 40], AcaNAP6 [SEQ. ID. NOS. 6 and 41], AcaNAP48
[SEQ. ID. NO. 42], AcaNAP23 [SEQ. ID. NO. 43], AcaNAP24 [SEQ. ID.
NO. 44], AcaNAP25 [SEQ. ID. NO. 45], AcaNAP44 [SEQ. ID. NO. 46],
AcaNAP31 [SEQ. ID. NO. 47], AduNAP4 [SEQ. ID. NO. 55], AceNAP5
[SEQ. ID. NO. 57], and AceNAP7 [SEQ. ID. NO. 58] have one NAP
domain and are preferred NAPs according to this embodiment. NAP
proteins AceNAP4 [SEQ. ID. NO. 62], AcaNAP45 [SEQ. ID. NO. 63],
AcaNAP47 [SEQ. ID. NO. 64], and AduNAP7 [SEQ. ID. NO. 65] have two
NAP domains and are preferred NAPs according to this
embodiment.
[0244] Preferred NAP proteins having anticoagulant activity,
according to all the embodiments recited above for this aspect of
the invention, can be derived from a nematode species. A preferred
nematode species is selected from the group consisting of
Ancylostoma caninum, Ancylostoma ceylanicum, Ancylostoma duodenale,
Necator americanus, and Heligomosomoides polygyrus. Particularly
preferred are NAP proteins AcaNAP5 [SEQ. ID. NO. 4 and 40], AcaNAP6
[SEQ. ID. NO. 6 and 41], AcaNAP48 [SEQ. ID. NO. 42], AcaNAP23 [SEQ.
ID. NO. 43], AcaNAP24 [SEQ. ID. NO. 44], AcaNAP25 [SEQ. ID. NO.
45], AcaNAP44 [SEQ. ID. NO. 46], AcaNAP45 [SEQ. ID. NO. 63],
AcaNAP47 [SEQ. ID. NO. 64], and AcaNAP31 [SEQ. ID. NO. 47] derived
from Ancylostoma caninum; AceNAP4 [SEQ. ID. NO. 62], AceNAP5 [SEQ.
ID. NO. 57], and AceNAP7 [SEQ. ID. NO. 58] derived from Ancylostoma
ceylanicum; and AduNAP7 [SEQ. ID. NO. 65] and AduNAP4 [SEQ. ID. NO.
55] derived from Ancylostoma duodenale.
[0245] This aspect of the invention also contemplates isolated
recombinant cDNA molecules encoding a protein having anticoagulant
activity, wherein the protein is defined according to each of the
embodiments recited above for isolated NAP protein having
anticoagulant activity. Preferred cDNAs according to this aspect
include AcaNAP5 [SEQ. ID. NO. 3], AcaNAP6 [SEQ. ID. NO. 5],
AcaNAP48 [SEQ. ID. NO. 38], AcaNAP23 [SEQ. ID. NO. 31], AcaNAP24
[SEQ. ID. NO. 32], AcaNAP25 [SEQ. ID. NO. 33], AcaNAP44 [SEQ. ID.
NO. 35], AcaNAP31 [SEQ. ID. NO. 34], AduNAP4 [SEQ. ID. NO. 12],
AceNAP5 [SEQ. ID. NO. 10], AceNAP7 [SEQ. ID. NO. 11], AceNAP4 [SEQ.
ID. NO. 9], AcaNAP45 [SEQ. ID. NO. 36], AcaNAP47 [SEQ. ID. NO. 37],
and AduNAP7 [SEQ. ID. NO. 13].
[0246] The anticoagulation activity of NAPs within this aspect of
the invention can be determined using protocols described herein.
Examples B and F present particulary useful methods for assessing a
NAP's anticoagulation activity. The procedures described for
detecting NAPs having fXa inhibitory activity (Examples A, C) and
fVIIa/TF inhibitory activity (Example E) also are useful in
evaluating a NAP's anticoagulation activity.
[0247] Oligonucleotides
[0248] Another aspect of this invention is an oligonucleotide
comprising a sequence selected from
1 YG109: TCAGACATGT-ATAATCTCAT-GTTGG, [SEQ. ID. NO. 88] YG103:
AAGGCATACC-CGGAGTGTGG-TG, [SEQ. ID. NO. 89] NAP-1.:
AAR-CCN-TGY-GAR-MGG-AAR-TGY, [SEQ. ID. NO. 90] and NAP-4.RC:
TW-RWA-NCC-NTC-YTT-RCA-NA- C-RCA. [SEQ. ID. NO. 91].
[0249] These oligonucleotide sequences hybridize to nucleic acid
sequences coding for NAP protein.
[0250] The isolated NAPs of the present invention include those
having variations in the disclosed amino acid sequence or
sequences, including fragments, naturally occurring mutations,
allelic variants, randomly generated artificial mutants and
intentional sequence variations, all of which conserve
anticoagulant activity. The term "fragments " refers to any part of
the sequence which contains fewer amino acids than the complete
protein, as for example, partial sequences excluding portions at
the amino-terminus, carboxy-terminus or between the amino-terminus
and carboxy-terminus of the complete protein.
[0251] The isolated NAPs of the present invention also include
proteins having a recombinant amino acid sequence or sequences
which conserve the anticoagulant activity of the NAP domain amino
acid sequence or sequences. Thus, as used herein, the phrase "NAP
protein" or the term "protein" when referring to a protein
comprising a NAP domain, means, without discrimination, native NAP
protein and NAP protein made by recombinant means. These
recombinant proteins include hybrid proteins, such as fusion
proteins, proteins resulting from the expression of multiple genes
within the expression vector, proteins resulting from expression of
multiple genes within the chromosome of the host cell, and may
include a polypeptide having anticoagulant activity of a disclosed
protein linked by peptide bonds to a second polypeptide. The
recombinant proteins also include variants of the NAP domain amino
acid sequence or sequences of the present invention that differ
only by conservative amino acid substitution. Conservative amino
acid substitutions are defined as "sets" in Table 1 of Taylor, W.
R., J. Mol. Biol., 188:233 (1986). The recombinant proteins also
include variants of the disclosed isolated NAP domain amino acid
sequence or sequences of the present invention in which amino acid
substitutions or deletions are made which conserve the
anticoagulant activity of the isolated NAP domain sequence or
sequences.
[0252] One preferred embodiment of the present invention is a
protein isolated by biochemical methods from the nematode,
Ancylostoma caninum, as described in Example 1. This protein
increases the clotting time of human plasma in the PT and aPTT
assays, contains one NAP domain, and is characterized by an
N-terminus having the amino acid sequence,
Lys-Ala-Tyr-Pro-Glu-Cys-Gly-Glu-Asn-Glu-Trp-Leu-Asp [SEQ. ID. NO.
92], and a molecular weight of about 8.7 kilodaltons to about 8.8
kilodaltons as determined by mass spectrometry.
[0253] Further preferred embodiments of the present invention
include the proteins having anticoagulant activity made by
recombinant methods from the cDNA library isolated from the
nematode, Ancylostoma caninum, for example, AcaNAP5 [SEQ. ID. NO. 4
or 40], AcaNAP6 [SEQ. ID. NO. 6 or 41], Pro-AcaNAP5 [SEQ. ID. NO.
7], Pro-AcaNAP6 [SEQ. ID. NO. 8], AcaNAP48 [SEQ. ID. NO. 42],
AcaNAP23 [SEQ. ID. NO. 43], AcaNAP24 [SEQ. ID. NO. 44], AcaNAP25
[SEQ. ID. NO. 45], AcaNAP44 [SEQ. ID. NO. 46], AcaNAP31 [SEQ. ID.
NO. 47], AcaNAP45 [SEQ. ID. NO. 63], AcaNAP47 [SEQ. ID. NO. 64],
and AcaNAPc2 [SEQ. ID. NO. 59]; isolated from the nematode,
Ancyclostoma ceylanium, for example, AceNAP4 [SEQ. ID. NO. 62],
AceNAP5 [SEQ. ID. NO. 57], and AceNAP7 [SEQ. ID. NO. 58]; isolated
from the nematode, Ancyclostoma duodenale, for example, AduNAP4
[SEQ. ID. NO. 55] and AduNAP7 [SEQ. ID. NO. 65]; isolated from the
nematode Heligmosmoides polygyrus, for example, HpoNAP5 [SEQ. ID.
NO. 60]; and the nematode Necator americanus, for example, NamNAP
[SEQ. ID. NO. 61]. The amino acid sequences of these proteins are
shown in FIGS. 11 and 16 and elsewhere. Each such preferred
embodiment increases the clotting time of human plasma in the PT
and aPTT assays and contains at least one NAP domain.
[0254] With respect to "isolated proteins", the proteins of the
present invention are isolated by methods of protein purification
well known in the art, or as disclosed below. They may be isolated
from a natural source, from a chemical mixture after chemical
synthesis on a solid phase or in solution such as solid-phase
automated peptide synthesis, or from a cell culture after
production by recombinant methods.
[0255] As described further hereinbelow, the present invention also
contemplates pharmaceutical compositions comprising NAP and methods
of using NAP to inhibit the process of blood coagulation and
associated thrombosis. Oligonucleotide probes useful for
identifying NAP nucleic acid in a sample also are within the
purview of the present invention, as described more fully
hereinbelow.
[0256] 1. NAP Isolated from Natural Sources.
[0257] The preferred isolated proteins (NAPs) of the present
invention may be isolated and purified from natural sources.
Preferred as natural sources are nematodes; suitable nematodes
include intestinal nematodes such as Ancylostoma caninum,
Ancylostoma ceylanicum, Ancylostoma duodenale, Necator americanus
and Heligmosomoides polygyrus. Especially preferred as a natural
source is the hematophagous nematode, the hookworm, Ancylostoma
caninum.
[0258] The preferred proteins of the present invention are isolated
and purified from their natural sources by methods known in the
biochemical arts. These methods include preparing a soluble extract
and enriching the extract using chromatographic methods on
different solid support matrices. Preferred methods of purification
would include preparation of a soluble extract of a nematode in
0.02 M Tris-HCl, pH 7.4 buffer containing various protease
inhibitors, followed by sequential chromatography of the extract
through columns containing Concanavalin-A Sepharose matrix, Poros20
HQ cation-ion exchange matrix, Superdex30 gel filtration matrix and
a C18 reverse-phase matrix. The fractions collected from such
chromatography columns may be selected by their ability to increase
the clotting time of human plasma, as measured by the PT and aPTT
assays, or their ability to inhibit factor Xa amidolytic activity
as measured in a colorimetric amidolytic assay using purified
enzyme, or by other methods disclosed in Examples A to F herein. An
example of a preferred method of purification of an isolated
protein of the present invention would include that as disclosed in
Example 1.
[0259] The preferred proteins of the present invention, when
purified from a natural source, such as Ancylostoma caninum, as
described, include those which contain the amino acid sequence:
Lys-Ala-Tyr-Pro-Glu-Cys-Gly-- Glu-Asn-Glu-Trp-Leu-Asp [SEQ. ID. NO.
92]. Especially preferred are the purified proteins having this
amino acid sequence at its amino terminus, such as shown in FIG. 2
(AcaNAP5 [SEQ. ID. NO. 4]) or FIG. 4 (AcaNAP6 [SEQ. ID. NO. 6]).
One preferred protein of the present invention was demonstrated to
have the amino acid sequence, Lys-Ala-Tyr-Pro-Glu-Cys-Gly-
-Glu-Asn-Glu-Trp-Leu-Asp [SEQ. ID. NO. 92] at its amino-terminus
and a molecular weight of 8.7 to 8.8 kilodaltons, as determined by
mass spectrometry.
[0260] 2. NAP Made by Chemical Synthesis.
[0261] The preferred isolated NAPs of the present invention may be
synthesized by standard methods known in the chemical arts.
[0262] The isolated proteins of the present invention may be
prepared using solid-phase synthesis, such as that described by
Merrifield, J. Amer. Chem. Soc., 85:2149 (1964) or other equivalent
methods known in the chemical arts, such as the method described by
Houghten in Proc. Natl. Acad. Sci., 82:5132 (1985).
[0263] Solid-phase synthesis is commenced from the C-terminus of
the peptide by coupling a protected amino acid or peptide to a
suitable insoluble resin. Suitable resins include those containing
chloromethyl, bromomethyl, hydroxylmethyl, aminomethyl, benzhydryl,
and t-alkyloxycarbonylhydrazide groups to which the amino acid can
be directly coupled.
[0264] In this solid phase synthesis, the carboxy terminal amino
acid, having its alpha amino group and, if necessary, its reactive
side chain group suitably protected, is first coupled to the
insoluble resin. After removal of the alpha amino protecting group,
such as by treatment with trifluoroacetic acid in a suitable
solvent, the next amino acid or peptide, also having its alpha
amino group and, if necessary, any reactive side chain group or
groups suitably protected, is coupled to the free alpha amino group
of the amino acid coupled to the resin. Additional suitably
protected amino acids or peptides are coupled in the same manner to
the growing peptide chain until the desired amino acid sequence is
achieved. The synthesis may be done manually, by using automated
peptide synthesizers, or by a combination of these.
[0265] The coupling of the suitably protected amino acid or peptide
to the free alpha amino group of the resin-bound amino acid can be
carried out according to conventional coupling methods, such as the
azide method, mixed anhydride method, DCC
(dicyclohexylcarbodiimide) method, activated ester method
(p-nitrophenyl ester or N-hydroxysuccinimide ester), BOP
(benzotriazole-1-yl-oxy-tris(diamino)phosphonium
hexafluorophosphate) method or Woodward reagent K method.
[0266] It is common in peptide synthesis that the protecting groups
for the alpha amino group of the amino acids or peptides coupled to
the growing peptide chain attached to the insoluble resin will be
removed under conditions which do not remove the side chain
protecting groups. Upon completion of the synthesis, it is also
common that the peptide is removed from the insoluble resin, and
during or after such removal, the side chain protecting groups are
removed.
[0267] Suitable protecting groups for the alpha amino group of all
amino acids and the omega amino group of lysine include
benzyloxycarbonyl, isonicotinyloxycarbonyl,
o-chlorobenzyloxycarbonyl, p-nitrophenyloxycarbonyl,
p-methoxyphenyloxycarbonyl, t-butoxycarbonyl, t-amyloxycarbonyl,
adamantyloxycarbonyl, 2-(4-biphenyl)-2-propyloxycarbon- yl,
9-fluorenylmethoxycarbonyl, methylsulfonylethoxylcarbonyl,
trifluroacetyl, phthalyl, formyl, 2-nitrophenylsulfphenyl,
diphenylphosphinothioyl, dimethylphosphinothioyl, and the like.
[0268] Suitable protecting groups for the carboxy group of aspartic
acid and glutamic acid include benzyl ester, cyclohexyl ester,
4-nitrobenzyl ester, t-butyl ester, 4-pyridylmethyl ester, and the
like.
[0269] Suitable protecting groups for the guanidino group of
arginine include nitro, p-toluenesulfonyl, benzyloxycarbonyl,
adamantyloxycarbonyl, p-methoxybenzenesulfonyl,
4-methoxy-2,6-dimethylben- zenesulfonyl,
1,3,5-trimethylphenylsulfonyl, and the like.
[0270] Suitable protecting groups for the thiol group of cysteine
include p-methoxybenzyl, triphenylmethyl, acetylaminomethyl,
ethylcarbamoyl, 4-methylbenzyl, 2,4,6-trimethylbenzyl, and the
like.
[0271] Suitable protecting groups for the hydroxy group of serine
include benzyl, t-butyl, acetyl, tetrahydropyranyl, and the
like.
[0272] The completed peptide may be cleaved from the resin by
treatment with liquid hydrofluoric acid containing one or more
thio-containing scavengers at reduced temperatures. The cleavage of
the peptide from the resin by such treatment will also remove all
side chain protecting groups from the peptide.
[0273] The cleaved peptide is dissolved in dilute acetic acid
followed by filtration, then is allowed to refold and establish
proper disulfide bond formation by dilution to a peptide
concentration of about 0.5 mM to about 2 mM in a 0.1 M acetic acid
solution. The pH of this solution is adjusted to about 8.0 using
ammonium hydroxide and the solution is stirred open to air for
about 24 to about 72 hours.
[0274] The refolded peptide is purified by chromatography,
preferably by high pressure liquid chromatography on a reverse
phase column, eluting with a gradient of acetonitrile in water
(also containing 0.1% trifluoroacetic acid), with the preferred
gradient running from 0 to about 80% acetonitrile in water. Upon
collection of fractions containing the pure peptide, the fractions
are pooled and lyophilized to the solid peptide.
[0275] 3. NAP Made by Recombinant Methods.
[0276] Alternatively, the preferred isolated NAPs of the present
invention may be made by recombinant DNA methods taught herein and
well known in the biological arts. Sambrook, J., Fritsch, E. F. and
Maniatis, T., Molecular Cloning, A Laboratory Manual, Second
Edition, volumes 1 to 3, Cold Spring Harbor Laboratory Press
(1989).
[0277] Recombinant DNA methods allow segments of genetic
information, DNA, from different organisms, to be joined together
outside of the organisms from which the DNA was obtained and allow
this hybrid DNA to be incorporated into a cell that will allow the
production of the protein for which the original DNA encodes.
[0278] Genetic information encoding a protein of the present
invention may be obtained from the genomic DNA or mRNA of an
organism by methods well known in the art. Preferred methods of
obtaining this genetic information include isolating mRNA from an
organism, converting it to its complementary DNA (cDNA),
incorporating the cDNA into an appropriate cloning vector, and
identifying the clone which contains the recombinant cDNA encoding
the desired protein by means of hybridization with appropriate
oligonucleotide probes constructed from known sequences of the
protein.
[0279] The genetic information in the recombinant cDNA encoding a
protein of the present invention may be ligated into an expression
vector, the vector introduced into host cells, and the genetic
information expressed as the protein for which it encodes.
[0280] (A) Preparation of cDNA Library.
[0281] Preferred natural sources of mRNA from which to construct a
cDNA library are nematodes which include intestinal nematodes such
as Ancylostoma caninum, Ancylostoma ceylanicum, Ancylostoma
duodenale, Necator americanus and Heligmosomoides polygyrus.
Especially preferred as a natural source of mRNA is the hookworm
nematode, Ancylostoma caninum.
[0282] Preferred methods of isolating mRNA encoding a protein of
the present invention, along with other mRNA, from an organism
include chromatography on poly U or poly T affinity gels.
Especially preferred methods of isolating the mRNA from nematodes
include the procedure and materials provided in the QuickPrep mRNA
Purification kit (Pharmacia).
[0283] Preferred methods of obtaining double-stranded cDNA from
isolated mRNA include synthesizing a single-stranded cDNA on the
mRNA template using a reverse transcriptase, degrading the RNA
hybridized to the cDNA strand using a ribonuclease (RNase), and
synthesizing a complementary DNA strand by using a DNA polymerase
to give a double-stranded cDNA. Especially preferred methods
include those wherein about 3 micrograms of mRNA isolated from a
nematode is converted into double-stranded cDNA making use of Avian
Myeloblastosis Virus reverse transcriptase, RNase H, and E. coli
DNA polymerase I and T4 DNA polymerase.
[0284] cDNA encoding a protein of the present invention, along with
the other cDNA in the library constructed as above, are then
ligated into cloning vectors. Cloning vectors include a DNA
sequence which accommodates the cDNA from the cDNA library. The
vectors containing the cDNA library are introduced into host cells
that can exist in a stable manner and provide a environment in
which the cloning vector is replicated. Suitable cloning vectors
include plasmids, bacteriophages, viruses and cosmids. Preferred
cloning vectors include the bacteriophages. Cloning vectors which
are especially preferred include the bacteriophage, lambda gt11
Sfi-Not vector.
[0285] The construction of suitable cloning vectors containing the
cDNA library and control sequences employs standard ligation and
restriction techniques which are well known in the art. Isolated
plasmids, DNA sequences or synthesized oligonucleotides are
cleaved, tailored and religated in the form desired.
[0286] With respect to restriction techniques, site-specific
cleavage of cDNA is performed by treating with suitable restriction
enzyme under conditions which are generally understood in the art,
and the particulars of which are specified by the manufacturer of
these commercially available restriction enzymes. For example, see
the product catalogs of New England Biolabs, Promega and Stratagene
Cloning Systems.
[0287] Generally, about 1 microgram of the cDNA is cleaved by
treatment in about one unit of a restriction enzyme in about 20
microliters of buffer solution. Typically, an excess of restriction
enzyme is used to ensure complete cleavage of the cDNA. Incubation
times of about 1 to 2 hours at about 37.degree. C. are usually
used, though exceptions are known. After each cleavage reaction,
the protein may be removed by extraction with phenol/chloroform,
optionally followed by chromatography over a gel filtration column,
such as Sephadex.RTM. G50. Alternatively, cleaved cDNA fragments
may be separated by their sizes by electrophoresis in
polyacrylamide or agarose gels and isolated using standard
techniques. A general description of size separations is found in
Methods of Enzymology, 65:499-560 (1980).
[0288] The restriction enzyme-cleaved cDNA fragments are then
ligated into a cloning vector.
[0289] With respect to ligation techniques, blunt-end ligations are
usually performed in about 15 to about 30 microliters of a pH 7.5
buffer comprising about 1 mM ATP and about 0.3 to 0.6 (Weiss) units
of T4 DNA ligase at about 14.degree. C. Intermolecular "sticky end"
ligations are usually performed at about 5 to 100 nanomolar
total-end DNA concentrations. Intermolecular blunt-end ligations
(usually employing about 10 to 30-fold molar excess of linkers) are
performed at about 1 micromolar total-end DNA concentrations.
[0290] (B) Preparation of cDNA Encoding NAP.
[0291] Cloning vectors containing the cDNA library prepared as
disclosed are introduced into host cells, the host cells are
cultured, plated, and then probed with a hybridization probe to
identify clones which contain the recombinant cDNA encoding a
protein of the present invention. Preferred host cells include
bacteria when phage cloning vectors are used. Especially preferred
host cells include E. coli strains such as strain Y1090.
[0292] Alternatively, the recombinant cDNA encoding a protein of
the present invention may be obtained by expression of such protein
on the outer surface of a filamentous phage and then isolating such
phage by binding them to a target protein involved in blood
coagulation.
[0293] An important and well known feature of the genetic code is
its redundancy--more than one triplet nucleotide sequence codes for
one amino acid. Thus, a number of different nucleotide sequences
are possible for recombinant cDNA molecules which encode a
particular amino acid sequence for a NAP of the present invention.
Such nucleotide sequences are considered functionally equivalent
since they can result in the production of the same amino acid
sequence in all organisms. Occasionally, a methylated variant of a
purine or pyrimidine may be incorporated into a given nucleotide
sequence. However, such methylations do not affect the coding
relationship in any way.
[0294] (1) Using Oligonucleotide Probes.
[0295] Hybridization probes and primers are oligonucleotide
sequences which are complementary to all or part of the recombinant
cDNA molecule that is desired. They may be prepared using any
suitable method, for example, the phosphotriester and
phosphodiester methods, described respectively in Narang, S. A. et
al., Methods in Enzymology, 68:90 (1979) and Brown, E. L. et al.,
Methods in Enzymology, 68:109 (1979), or automated embodiments
thereof. In one such embodiment, diethylphosphoramidites are used
as starting materials and may be synthesized as described by
Beaucage et al., Tetrahedron Letters, 22:1859-1862 (1981). One
method for synthesizing oligonucleotides on a modified solid
support is described in U.S. Pat. No. 4,458,066. Probes differ from
primers in that they are labelled with an enzyme, such as
horseradish peroxidase, or radioactive atom, such as .sup.32P, to
facilitate their detection. A synthesized probe is radiolabeled by
nick translation using E. coli DNA polymerase I or by end labeling
using alkaline phosphatase and T4 bacteriophage polynucleotide
kinase.
[0296] Preferred hybridization probes include oligonucleotide
sequences which are complementary to a stretch of the
single-stranded cDNA encoding a portion of the amino acid sequence
of a NAP purified from a nematode, such as the hookworm,
Ancylostoma caninum. For example, a portion of the amino acid
sequence shown in FIG. 2 (AcaNAP5) [SEQ. ID. NO. 4] or FIG. 4
(AcaNAP6 [SEQ. ID. NO. 6]) can be used. Especially preferred
hybridization probes include those wherein their oligonucleotide
sequence is complementary to the stretch of the single-stranded
cDNA encoding the amino acid sequence:
Lys-Ala-Tyr-Pro-Glu-Cys-Gly-Glu-Asn-Glu-Trp [SEQ. ID. NO. 93]. Such
hybridization probes include the degenerate probe having the
oligonucleotide sequence: AAR GCi TAY CCi GAR TGY GGi GAR AAY GAR
TGG [SEQ. ID. NO. 94], wherein R is A or G, Y is T or C, and i is
inosine. A preferred recombinant cDNA molecule encoding a protein
of the present invention is identified by its ability to hybridize
to this probe.
[0297] Preferred hybridization probes also include the pair NAP-1
[SEQ. ID. NO. 90] and NAP-4.RC [SEQ. ID. NO. 91], and the pair
YG109 [SEQ. ID. NO. 88] and YG103 [SEQ. ID. NO. 89], both of which
are described in Examples 13 and 12, respectively.
[0298] Upon identification of the clone containing the desired
cDNA, amplification is used to produce large quantities of a gene
encoding a protein of the present invention in the form of a
recombinant cDNA molecule.
[0299] Preferred methods of amplification include the use of the
polymerase chain reaction (PCR). See, e.g., PCR Technology, W. H.
Freeman and Company, New York (Edit. Erlich, H. A. 1992). PCR is an
in vitro amplification method for the synthesis of specific DNA
sequences. In PCR, two oligonucleotide primers that hybridize to
opposite strands and flank the region of interest in the cDNA of
the clone are used. A repetitive series of cycles involving cDNA
denaturation into single strands, primer annealing to the
single-stranded cDNA, and the extension of the annealed primers by
DNA polymerase results in number of copies of cDNA, whose termini
are defined by the 5-ends of the primers, approximately doubling at
every cycle. Ibid., p.1. Through PCR amplification, the coding
domain and any additional primer encoded information such as
restriction sites or translational signals (signal sequences, start
codons and/or stop codons) of the recombinant cDNA molecule to be
isolated is obtained.
[0300] Preferred conditions for amplification of cDNA include those
using Taq polymerase and involving 30 temperature cycles of: 1
minute at 95.degree. C.; 1 minute at 50.degree. C.; 1.5 minutes at
72.degree. C. Preferred primers include the oligo(dT)-NotI primer,
AATTCGCGGC CGC(T)15 [SEQ. ID. NO. 95], obtained from Promega Corp.
in combination with either (i) the degenerate primer having the
oligonucleotide sequence: AAR GCi TAY CCi GAR TGY GGi GAR AAY GAR
TGG [SEQ. ID. NO. 94], wherein R is A or G, Y is T or C, and i is
inosine, or (ii) the lambda gt11 primer #1218, GGTGGCGACG
ACTCCTGGAG CCCG [SEQ. ID. NO. 96], obtained from New England
Biolabs.
[0301] The nucleic acid sequence of a recombinant cDNA molecule
made as disclosed is determined by methods based on the dideoxy
method of Sanger, F. et al, Proc. Natl. Acad. Sci. USA, 74:5463
(1977) as further described by Messing, et al., Nucleic Acids Res.,
2:309 (1981).
[0302] Preferred recombinant cDNA molecules made as disclosed
include those having the nucleic acid sequences of FIGS. 1, 3, 7,
9, 13, and 14.
[0303] (2) Using NAP cDNAs as Probes.
[0304] Also especially preferred as hybridization probes are
oligonucleotide sequences encoding substantially all of the amino
acid sequence of a NAP purified from the nematode, the hookworm,
Ancylostoma caninum. Especially preferred probes include those
derived from the AcaNAP5 and AcaNAP6 genes and having the following
nucleic acid sequences (AcaNAP5 gene):
2 AAG GCA TAC CCG GAG TGT GGT GAG [SEQ. ID. NO. 1] AAT GAA TGG CTC
GAC GAC TGT GGA ACT CAG AAG CCA TGC GAG GCC AAG TGC AAT GAG GAA CCC
CCT GAG GAG GAA GAT CCG ATA TGC CGC TCA CGT GGT TGT TTA TTA CCT CCT
GCT TGC GTA TGC AAA GAC GGA TTC TAC AGA GAC ACG GTG ATC GGC GAC TGT
GTT AGG GAA GAA GAA TGC GAC CAA CAT GAG ATT ATA CAT GTC TGA, or
FIG. 3 (AcaNAP6 gene): AAG GCA TAC CCG GAG TGT GGT GAG [SEQ. ID.
NO. 2] AAT GAA TGG CTC GAC GTC TGT GGA ACT AAG AAG CCA TGC GAG GCC
AAG TGC AGT GAG GAA GAG GAG GAA GAT CCG ATA TGC CGA TCA TTT TCT TGT
CCG GGT CCC GCT GCT TGC GTA TGC GAA GAC GGA TTC TAC AGA GAC ACG GTG
ATC GGC GAC TGT GTT AAG GAA GAA GAA TGC GAC CAA CAT GAG ATT ATA CAT
GTC TGA.
[0305] Preferred hybridization probes also include sequences
encoding a substantial part of the amino acid sequence of a NAP,
such as the PCR fragment generated with the primer couple NAP-1
[SEQ. ID. NO. 90] and NAP-4.RC [SEQ. ID. NO. 91] as described in
Example 13.
[0306] (3) Using Phage Display.
[0307] Disclosed herein is a method to select cDNAs encoding the
proteins of the present invention from whole cDNA libraries making
use of filamentous phage display technology. Current display
technology with filamentous phage relies on the in-frame insertion
of coding regions of interest into gene 3 or gene 8 which code for
the attachment protein and major coat protein of the phage,
respectively. Those skilled in the art will recognize that various
difficulties are inherent in performing this with a vast mixture of
cDNAs of unknown sequence and that the most practical way to obtain
functional display of cDNA products would consist of fusing the
cDNAs through their 5'-end. Indeed, cDNA libraries of sufficient
size may contain several cDNAs which derive from the same mRNA but
which are 5'-terminally truncated at various positions such that
some of them may be expressed as fusion products. A strategy along
this line, which relies on the ability of the leucine zippers Jun
and Fos to form heterodimers was recently described. See, Crameri,
R. and Suter, M., Gene, 137:69-75 (1993).
[0308] We have found a novel alternative and direct way to
convalently link cDNA gene products to the phage surface; the
finding is based on the observation that proteins fused to the
C-terminus of phage coat protein 6 can be functionally displayed.
This observation has led to the development of a phagemid system as
described herein which allows the expression of functionally
displayed cDNA products, which in turn permits the
affinity-selection of phage particles which contain the cDNA
required for the production of the displayed cDNA product. This
system provides the basis for the isolation of cDNAs which encode a
protein of the present invention. Once isolated, recombinant cDNA
molecules containing such cDNA can be used for expression of the
proteins of the present invention in other expression systems. The
recombinant cDNA molecules made in this way are considered to be
within the scope of the present invention.
[0309] Recombinant cDNA molecules of the present invention are
isolated by preparing a cDNA library from a natural source (as for
example, a nematode such as a hookworm), ligating this cDNA library
into appropriate phagemid vectors, transforming host cells with
these vectors containing the cDNAs, culturing the host cells,
infecting the transformed cells with an appropriate helper phage,
separating phage from the host cell culture, separating phage
expressing a protein of the present invention on its surface,
isolating these phage, and isolating a recombinant cDNA molecule
from such phage.
[0310] The phagemid vectors are constructed using the pUC119
expression vector described by Vieira, J. and Messing, J., Methods
in Enzymology, 153:3-11 (1987). The filamentous phage gene 6
encoding a surface protein of the phage is modified on its 5' and
3' ends by the addition of HindIII and SfiI restriction sites,
respectively, by use of three forward primers and one backward
primer using PCR. This results in three DNA fragments which are
further modified by addition to their 3' ends of NotI and BamHI
restriction sites by PCR. After separate digestion of the three DNA
fragments with HindIII and BamHI, the three DNA fragments are
ligated into the pUC119 to give pDONG61, pDONG62 and pDONG63
expression vectors. These vectors permit the insertion of cDNA as
SfiI-NotI fragments into them.
[0311] cDNA libraries are prepared from natural sources, such as
nematodes, as described in Examples 2, 9, and 13. Preferred
nematodes from which to make such libraries include the intestinal
nematodes such as Ancylostoma caninum, Ancylostoma ceylanicum,
Ancylostoma duodenale, Necator americanus and Heligmosomoides
polygyrus.
[0312] A cDNA library as SfiI-NotI fragments may be directly
directionally ligated into the phagemid vectors pDONG61, pDONG62
and pDONG63. Alternatively, a cDNA library which has been ligated
into the lambda gt11 phage vector as described in Example 2 can be
recovered by PCR, followed by isolation with electrophoresis and
then directional ligation into these vectors. In the latter
approach, preferred conditions for PCR use Taq polymerase; the
primers, lambda gt11 primer #1218 having the sequence GGTGGCGACG
ACTCCTGGAG CCCG (New England Biolabs, Beverly, Mass., USA) [SEQ.
ID. NO. 96] and the oligo(dT)-NotI primer having the sequence,
AATTCGCGGC CGC(T).sub.15, (Promega Corp.) [SEQ. ID. NO. 95]; and 20
temperature cycles of 1 minute at 95.degree. C., 1 minute at
50.degree. C., and 3 minutes at 72.degree. C., followed by 10
minutes at 65.degree. C.
[0313] Host cells are transformed with the pDONG expression vectors
containing a cDNA library. Preferred host cells include E. coli
strains, with strain TG1 being especially preferred. Preferred
methods for the transformation of E. coli host cells include
electroporation.
[0314] The transformed cells are cultured at 37.degree. C. in LB
medium supplemented with 1% glucose and 100 micrograms/ml
carbenicillin until the optical absorbance at 600 nm reaches the
value of 0.5 and then are infected with VCSM13 helper phage
(Stratagene) at a multiplicity of infection (moi) of 20.
[0315] The phage are separated from the culture by centrifugation,
then are purified by precipitations with polyethylene glycol/sodium
chloride.
[0316] The phage which express a NAP of the present invention on
their surface are isolated by taking advantage of the ability of
the NAP to bind to a target protein involved in blood coagulation,
for example, Factor Xa.
[0317] Preferred methods of isolating such phage include a method
comprising the steps of:
[0318] (1) combining a solution of factor Xa labelled to biotin
with a solution of such phage;
[0319] (2) incubating this mixture;
[0320] (3) contacting a solid phase labelled with streptavidin with
this mixture;
[0321] (4) incubating the solid phase with the mixture;
[0322] (5) removing the solid phase from the mixture and contacting
the solid phase with buffer to remove unbound phage;
[0323] (6) contacting the solid phase with a second buffer to
remove the bound phage from the solid phase;
[0324] (7) isolating such phage;
[0325] (8) transforming host cells with such phage;
[0326] (9) culturing the transformed host cells;
[0327] (10) infecting transformed host cells with VCSM13 helper
phage;
[0328] (11) isolating the phage from the host cell culture; and
[0329] (12) repeating steps (1) to (11) four more times.
[0330] An especially preferred method of isolating such phage
include the method as detailed in Example 10.
[0331] Single-stranded DNA was prepared from the isolated phages
and their inserts 3' to the filamentous phage gene 6 sequenced.
[0332] FIG. 9 depicts the recombinant cDNA molecule, AcaNAPc2,
isolated by the phage display method. The deduced amino acid
sequence of the protein of the present invention encoded by
AcaNAPc2 is also shown in this figure.
[0333] (C) Preparation of Recombinant NAP.
[0334] The recombinant cDNA molecules of the present invention when
isolated as disclosed are used to obtain expression of the NAPs of
the present invention. Generally, a recombinant cDNA molecule of
the present invention is incorporated into an expression vector,
this expression vector is introduced into an appropriate host cell,
the host cell is cultured, and the expressed protein is
isolated.
[0335] Expression vectors are DNA sequences that are required for
the transcription of cloned copies of genes and translation of
their mRNAs in an appropriate host. These vectors can express
either procaryotic or eucaryotic genes in a variety of cells such
as bacteria, yeast, mammalian, plant and insect cells. Proteins may
also be expressed in a number of virus systems.
[0336] Suitably constructed expression vectors contain an origin of
replication for autonomous replication in host cells, or are
capable of integrating into the host cell chromosomes. Such vectors
will also contain selective markers, a limited number of useful
restriction enzyme sites, a high copy number, and strong promoters.
Promoters are DNA sequences that direct RNA polymerase to bind to
DNA and initiate RNA synthesis; strong promoters cause such
initiation at high frequency. The preferred expression vectors of
the present invention are operatively linked to a recombinant cDNA
molecule of the present invention, i.e., the vectors are capable
directing both replication of the attached recombinant cDNA
molecule and expression of the protein encoded by the recombinant
cDNA molecule. Expression vectors may include, but are not limited
to cloning vectors, modified cloning vectors and specifically
designed plasmids or viruses.
[0337] Suitable host cells for expression of the proteins of the
present invention include bacteria, yeast, mammalian, plant and
insect cells. With each type of cell and species therein certain
expression vectors are appropriate as will be disclosed below.
[0338] Procaryotes may be used for expression of the proteins of
the present invention. Suitable bacteria host cells include the
various strains of E. coli, Bacillus subtilis, and various species
of Pseudomonas. In these systems, plasmid vectors which contain
replication sites and control sequences derived from species
compatible with the host are used. Suitable vectors for E. coli are
derivatives of pBR322, a plasmid derived from an E. coli species by
Bolivar et al., Gene, 2:95 (1977). Common procaryotic control
sequences, which are defined herein to include promoters for
transcription, initiation, optionally with an operator, along with
ribosome binding site sequences, include the beta-lactamase and
lactose promoter systems (Chang et al., Nature, 198:1056 (1977)),
the tryptophan promoter system (Goeddel et al., Nucleic Acids Res.,
8:4057 (1980)) and the lambda-derived-P.sub.L promoter and N-gene
ribosome binding site (Shimatake et al., Nature, 292:128 (1981)).
However, any available promoter system compatible with procaryotes
can be used. Preferred procaryote expression systems include E.
coli and their expression vectors.
[0339] Eucaryotes may be used for expression of the proteins of the
present invention. Eucaryotes are usually represented by the yeast
and mammalian cells. Suitable yeast host cells include
Saccharomyces cerevisiae and Pichia pastoris. Suitable mammalian
host cells include COS and CHO (chinese hamster ovary) cells.
[0340] Expression vectors for the eucaryotes are comprised of
promoters derived from appropriate eucaryotic genes. Suitable
promoters for yeast cell expression vectors include promoters for
synthesis of glycolytic enzymes, including those for
3-phosphoglycerate kinase gene in Saccharomyces cerevisiae (Hitzman
et al., J. Biol. Chem., 255:2073 (1980)) and those for the
metabolism of methanol as the alcohol oxidase gene in Pichia
pastoris (Stroman et al., U.S. Pat. Nos. 4,808,537 and 4,855,231).
Other suitable promoters include those from the enolase gene
(Holland, M. J. et al., J. Biol. Chem., 256:1385 (1981)) or the
Leu2 gene obtained from YEp13 (Broach, J. et al., Gene, 8:121
(1978)).
[0341] Preferred yeast expression systems include Pichia pastoris
and their expression vectors. NAP-encoding cDNAs expressed in
Pichia pastoris optionally may be mutated to encode a NAP protein
that incorporates a proline residue at the C-terminus. In some
instances the NAP protein is expressed at a higher level and can be
more resistant to unwanted proteolysis. One such cDNA, and its
expression in Pichia pastoris, is described in Example 17.
[0342] Suitable promoters for mammalian cell expression vectors
include the early and late promoters from SV40 (Fiers, et al.,
Nature, 273:113 (1978)) or other viral promoters such as those
derived from polyoma, adenovirus II, bovine papilloma virus or
avian sarcoma viruses. Suitable viral and mammalian enhancers may
also be incorporated into these expression vectors.
[0343] Suitable promoters for plant cell expression vectors include
the nopaline synthesis promoter described by Depicker, A. et al.,
Mol. Appl. Gen., 1:561 (1978).
[0344] Suitable promoters for insect cell expression vectors
include modified versions of the system described by Smith et al.,
U.S. Pat. No. 4,745,051. The expression vector comprises a
baculovirus polyhedrin promoter under whose control a cDNA molecule
encoding a protein can be placed.
[0345] Host cells are transformed by introduction of expression
vectors of the present invention into them. Transformation is done
using standard techniques appropriate for each type of cell. The
calcium treatment employing calcium chloride described in Cohen, S.
N., Proc. Natl. Acad. Sci. USA, 69:2110 (1972), or the RbCl method
described in Maniatis et al., Molecular Cloning: A Laboratory
Manual, p. 254, Cold Spring Harbor Press (1982) is used for
procaryotes or other cells which contain substantial cell wall
barriers. The transformation of yeast is carried out as described
in Van Solingen, P. et al., J. Bacter., 130:946 (1977) and Hsiao,
C. L. et al., Proc. Natl. Acad. Sci. USA, 76:3829 (1979). Mammalian
cells without much cell wall are transformed using the calcium
phosphate procedure of Graham and van der Eb, Virology, 52:546
(1978). Plant cells are transformed by infection with Agrobacterium
tumefaciens as described in Shaw, C. et al, Gene, 23:315 (1983).
Preferred methods of transforming E. coli and Pichia pastoris with
expression vectors include electroporation.
[0346] Transformed host cells are cultured under conditions, such
as type of media, temperature, oxygen content, fluid motion, etc.,
well known in the biological arts.
[0347] The recombinant proteins of the present invention are
isolated from the host cell or media by standard methods well known
in the biochemical arts, which include the use of chromatography
methods. Preferred methods of purification would include sequential
chromatography of an extract through columns containing Poros20 HQ
anion-ion exchange matrix or Poros20 HS cation exchange matrix,
Superdex30 gel filtration matrix and a C18 reverse-phase matrix.
The fractions collected after one such chromatography column may be
selected by their ability to increase the clotting time of human
plasma, as measured by the PT and aPTT assays, or their ability to
inhibit factor Xa amidolytic activity as measured in a colorimetric
assay, or demonstration of activity in any of the other assays
disclosed herein. Examples of preferred methods of purification of
a recombinant protein of the present invention are disclosed in
Examples 3, 4, 6, 8, 14 and 15.
[0348] 4. Methods of using NAP.
[0349] In one aspect, the present invention includes methods of
collecting mammalian plasma such that clotting of said plasma is
inhibited, comprising adding to a blood collection tube an amount
of a protein of the present invention sufficient to inhibit the
formation of a clot when mammalian blood is drawn into the tube,
adding mammalian blood to said tube, separating the red blood cells
from the mammalian plasma, and collecting the mammalian plasma.
[0350] Blood collection tubes include stoppered test tubes having a
vacuum therein as a means to draw blood obtained by venipuncture
into the tubes. Preferred test tubes include those which are made
of borosilicate glass, and have the dimensions of, for example,
10.25.times.47 mm, 10.25.times.50 mm, 10.25.times.64 mm,
10.25.times.82 mm, 13.times.75 mm, 13.times.100 mm, 16.times.75 mm,
16.times.100 mm or 16.times.125 mm. Preferred stoppers include
those which can be easily punctured by a blood collection needle
and which when placed onto the test tube provide a seal sufficient
to prevent leaking of air into the tube.
[0351] The proteins of the present invention are added to the blood
collection tubes in a variety of forms well known in the art, such
as a liquid composition thereof, a solid composition thereof, or a
liquid composition which is lyophilized to a solid in the tube. The
amount added to such tubes is that amount sufficient to inhibit the
formation of a clot when mammalian blood is drawn into the tube.
The proteins of the present invention are added to blood collection
tubes in such amounts that, when combined with 2 to 10 ml of
mammalian blood, the concentration of such proteins will be
sufficient to inhibit clot formation. Typically, this effective
concentration will be about 1 to 10,000 nM, with 10 to 1000 nM
being preferred. Alternatively, the proteins of the present
invention may be added to such tubes in combination with other
clot-inhibiting additives, such as heparin salts, EDTA salts,
citrate salts or oxalate salts.
[0352] After mammalian blood is drawn into a blood collection tube
containing either a protein of the present invention or the same in
combination with other clot-inhibiting additives, the red blood
cells are separated from the mammalian plasma by centrifugation.
The centrifugation is performed at g-forces, temperatures and times
well known in the medical arts. Typical conditions for separating
plasma from red blood cells include centrifugation at a centrifugal
force of about 100.times.g to about 1500.times.g, at a temperatures
of about 5 to about 25.degree. C., and for a time of about 10 to
about 60 minutes.
[0353] The mammalian plasma may be collected by pouring it off into
a separate container, by withdrawing it into a pipette or by other
means well known to those skilled in the medical arts.
[0354] In another aspect, the present invention includes methods
for preventing or inhibiting thrombosis (clot formation) or blood
coagulation in a mammal, comprising administering to said mammal a
therapeutically effective amount of a protein or a pharmaceutical
composition of the present invention.
[0355] The proteins or pharmaceutical compositions of the present
invention are administered in vivo, ordinarily in a mammal,
preferably in a human. In employing them in vivo, the proteins or
pharmaceutical compositions can be administered to a mammal in a
variety of ways, including orally, parenterally, intravenously,
subcutaneously, intramuscularly, colonically, rectally, nasally or
intraperitoneally, employing a variety of dosage forms.
Administration is preferably parenteral, such as intravenous on a
daily basis. Alternatively, administration is preferably oral, such
as by tablets, capsules or elixers taken on a daily basis.
[0356] In practicing the methods of the present invention, the
proteins or pharmaceutical compositions of the present invention
are administered alone or in combination with one another, or in
combination with other therapeutic or in vivo diagnostic
agents.
[0357] As is apparent to one skilled in the medical art, a
therapeutically effective amount of the proteins or pharmaceutical
compositions of the present invention will vary depending upon the
age, weight and mammalian species treated, the particular proteins
employed, the particular mode of administration and the desired
affects and the therapeutic indication. Because these factors and
their relationship to determining this amount are well known in the
medical arts, the determination of therapeutically effective dosage
levels, the amount necessary to achieve the desired result of
preventing thrombosis, will be within the ambit of one skilled in
these arts.
[0358] Typically, administration of the proteins or pharmaceutical
composition of the present invention is commenced at lower dosage
levels, with dosage levels being increased until the desired effect
of preventing in vivo thrombosis is achieved which would define a
therapeutically effective amount. For the proteins of the present
invention, alone or as part of a pharmaceutical composition, such
doses are between about 0.01 mg/kg and 100 mg/kg body weight,
preferably between about 0.01 and 10 mg/kg, body weight.
[0359] 5. Utility.
[0360] Proteins of the present invention when made and selected as
disclosed are useful as potent inhibitors of blood coagulation in
vitro and in vivo. As such, these proteins are useful as in vitro
diagnostic reagents to prevent the clotting of blood and are also
useful as in vivo pharmaceutical agents to prevent or inhibit
thrombosis or blood coagulation in mammals.
[0361] The proteins of the present invention are useful as in vitro
diagnostic reagents for inhibiting clotting in blood drawing tubes.
The use of stoppered test tubes having a vacuum therein as a means
to draw blood obtained by venipuncture into the tube is well known
in the medical arts. Kasten, B. L., "Specimen Collection",
Laboratory Test Handbook, 2nd Edition, Lexi-Comp Inc., Cleveland
pp. 16-17 (Edits. Jacobs, D. S. et al. 1990). Such vacuum tubes may
be free of clot-inhibiting additives, in which case, they are
useful for the isolation of mammalian serum from the blood. They
may alternatively contain clot-inhibiting additives (such as
heparin salts, EDTA salts, citrate salts or oxalate salts), in
which case, they are useful for the isolation of mammalian plasma
from the blood. The proteins of the present invention are potent
inhibitors of blood clotting and as such, can be incorporated into
blood collection tubes to prevent clotting of the mammalian blood
drawn into them.
[0362] The proteins of the present invention are used alone, in
combination of other proteins of the present invention, or in
combination with other known inhibitors of clotting, in the blood
collection tubes, for example, with heparin salts, EDTA salts,
citrate salts or oxalate salts.
[0363] The amount to be added to such tubes, or effective amount,
is that amount sufficient to inhibit the formation of a blood clot
when mammalian blood is drawn into the tube. The proteins of the
present invention are added to blood collection tubes in such
amounts that, when combined with 2 to 10 ml of mammalian blood, the
concentration of such proteins will be sufficient to inhibit the
formation of blood clots. Typically, this effective amount is that
required to give a final concentration in the blood of about 1 to
10,000 nM, with 10 to 1000 nM being preferred.
[0364] The proteins of the present invention may also be used to
prepare diagnostic compositions. In one embodiment, diagnostic
compositions are prepared by dissolving the proteins of the present
invention into diagnostically acceptable carriers, which carriers
include phosphate buffered saline (0.01 M sodium phosphate+0.15 M
sodium chloride, pH 7.2 or Tris buffered saline (0.05 M
Tris-HCl+0.15 M sodium chloride, pH 8.0). In another embodiment,
the proteins of the present invention may be blended with other
solid diagnostically acceptable carriers by methods well known in
the art to provide solid diagnostic compositions. These carriers
include buffer salts.
[0365] The addition of the proteins of the present invention to
blood collection tubes may be accomplished by methods well known in
the art, which methods include introduction of a liquid diagnostic
composition thereof, a solid diagnostic composition thereof, or a
liquid diagnostic composition which is lyophilized in such tubes to
a solid plug of a solid diagnostic composition.
[0366] The use of blood collection tubes containing the diagnostic
compositions of the present invention comprises contacting a
effective amount of such diagnostic composition with mammalian
blood drawn into the tube. Typically, when a sample of 2 to 10 ml
of mammalian blood is drawn into a blood collection tube and
contacted with such diagnostic composition therein; the effective
amount to be used will include those concentrations of the proteins
formulated as a diagnostic composition which in the blood sample
are sufficient to inhibit the formation of blood clots. Preferred
effective concentrations would be about 1 to 10,000 nM, with 10 to
1000 nM being especially preferred.
[0367] According to an alternate aspect of our invention, the
proteins of the present invention are also useful as pharmaceutical
agents for preventing or inhibiting thrombosis or blood coagulation
in a mammal. This prevention or inhibition of thrombosis or blood
coagulation includes preventing or inhibiting abnormal
thrombosis.
[0368] Conditions characterized by abnormal thrombosis are well
known in the medical arts and include those involving the arterial
and venous vasculature of mammals. With respect to the coronary
arterial vasculature, abnormal thrombosis (thrombus formation)
characterizes the rupture of an established atherosclerotic plaque
which is the major cause of acute myocardial infarction and
unstable angina, and also characterizes the occlusive coronary
thrombus formation resulting from either thrombolytic therapy or
percutaneous transluminal coronary angioplasty (PTCA). With respect
to the venous vasculature, abnormal thrombosis characterizes the
condition observed in patients undergoing major surgery in the
lower extremities or the abdominal area who often suffer from
thrombus formation in the venous vasculature resulting in reduced
blood flow to the affected extremity and a predisposition for
pulmonary embolism. Abnormal thrombosis further characterizes
disseminated intravascular coagulopathy which commonly occurs
within both vascular systems during septic shock, certain viral
infections and cancer, a condition wherein there is rapid
consumption of coagulation factors and systemic coagulation which
results in the formation of life-threatening thrombi occurring
throughout the microvasculature leading to widespread organ
failure.
[0369] The NAP proteins of the present invention also are useful
immunogens against which antibodies are raised. Antibodies, both
monoclonal and polyclonal, directed to a NAP are useful for
diagnostic purposes and for the identification of concentration
levels of NAP in various biological fluids. Immunoassay utilizing
these antibodies may be used as a diagnostic test, such as to
detect infection of a mammalian host by a parasitic worm or to
detect NAP from a parasitic worm in a tissue of the mammalian host.
Also, such immunoassays may be used in the detection and isolation
of NAP from tissue homogenates, cloned cells and the like.
[0370] NAP can be used, with suitable adjuvants, as a vaccine
against parasitic worm infections in mammals. Immunization with NAP
vaccine may be used in both the prophylaxis and therapy of
parasitic infections. Disease conditions caused by parasitic worms
may be treated by administering to an animal infected with these
parasites anti-NAP antibody.
[0371] NAP proteins of this invention having serine protease
inhibitory activity also are useful in conditions or assays where
the inhibition of serine protease is desired. For example, NAP
proteins that inhibit the serine protease trypsin or elastase are
useful for treatment of acute pancreatitis or acute inflammatory
response mediated by leukocytes, respectively.
[0372] The recombinant cDNA molecules encoding the proteins of the
present invention are useful in one aspect for isolating other
recombinant cDNA molecules which also encode the proteins of the
present invention. In another aspect, they are useful for
expression of the proteins of the present invention in host
cells.
[0373] The nucleotide probes of the present invention are useful to
identify and isolate nucleic acid encoding NAPs from nematodes or
other organisms. Additionally, the nucleotide probes are useful
diagnostic reagents to detect the presence of nematode-encoding
nucleic acid in a sample, such as a bodily fluid or tissue from a
mammal suspected of infection by nematode. The probes can be used
directly, with appropriate label for detection, to detect the
presence of nematode nucleic acid, or can be used in a more
indirect manner, such as in a PCR-type reaction, to amplify
nematode nucleic acid that may be present in the sample for
detection. The conditions of such methods and diagnostic assays are
readily available in the art.
[0374] To assist in understanding, the present invention will now
be be further illustrated by the following examples. These examples
as they relate to this invention should not be construed as
specifically limiting the invention and such variations of the
invention, now known or later developed, which would be within the
purview of one skilled in the art are considered to fall within the
scope of the invention as described herein and hereinafter
claimed.
EXAMPLES
Example 1
[0375] Isolation of Novel Anticoagulant Protein (NAP) from
Ancylostoma caninum.
[0376] (A) Preparation of the Ancylostoma caniumum Lysate.
[0377] Frozen canine hookworms, Ancylostoma caninum, were obtained
from Antibody Systems (Bedford, Tex.). Hookworms were stored at
-80.degree. C. until used for homogenate.
[0378] Hookworms were frozen in liquid nitrogen and ground in a
mortar followed by a homogenization on ice in homogenization buffer
using a PotterS homogenizer with a teflon piston (B. Braun
Melsungen AG, Germany). The homogenization buffer contained: 0.02 M
Tris-HCl pH 7.4, 0.05 M NaCl, 0.001 M MgCl.sub.2, 0.001 M
CaCl.sub.2, 1.0.times.10.sup.-5 M E-64 protease inhibitor
(Boehringer Mannheim, Germany), 1.0.times.10.sup.-5 M pepstatin A
(isovaleryl-Val-Val-4-amino-3-hydroxy-6-
-methyl-heptanoyl-Ala-4-amino-3-hydroxy-6-methylheptanoic acid, ICN
Biomedicals, CA), 1.0.times.10.sup.-5 M chymostatin (Boehringer),
1.0.times.10.sup.-5 M leupeptin (ICN), 5.times.10.sup.-5 M AEBSF
(4-(2-aminoethyl)-benzenesulfonyl fluoride, ICN), and 5% (v/v)
glycerol. Approximately 4 ml of homogenization buffer was used to
homogenize each gram of frozen worms (approximately 500 worms).
Insoluble material was pelleted by two sequential centrifugation
steps: 19,000.times.g.sub.max at 4.degree. C. for 30 minutes
followed by 110,000.times.g.sub.max at 4.degree. C. for 40 minutes.
The supernatant solution was clarified by passage through a 0.45
micrometer cellulose acetate filter (Corning, N.Y.) to give
Ancylostoma caniumum lysate.
[0379] (B) Concanavalin A Sepharose Chromatography.
[0380] Ancylostoma caniumum lysate (100 ml) was adsorbed onto 22 ml
of Concanavalin A Sepharose (Pharmacia, Sweden) pre-equilibrated
with Con A buffer (0.02 M Tris-HCl, pH 7.4, 1 M NaCl, 0.002 M
CaCl.sub.2) by loading it onto a 1.6.times.11 cm column of this gel
at a flow rate of 3 ml/minute (90 cm/hour). The column was at
ambient temperature while the reservoir of lysate was maintained at
ice bath temperature throughout the procedure. The column was
subsequently washed with 2 column volumes of Con A buffer. The
column flow-through and wash were collected (approximately 150 ml)
and stored at -80.degree. C. until further processing was done.
[0381] (C) Anion-Exchange Chromatography.
[0382] The flow-through and wash of the Concanavalin A Sepharose
column was buffered by adding solid sodium acetate to a final
concentration of 12.5 mM. The conductivity was reduced by dilution
with milliQ water and the pH was adjusted with HCl to pH 5.3. The
precipitate formed during pH adjustment was pelleted by
centrifugation 15,000.times.g.sub.max at 4.degree. C. for 15
minutes. The supernatant solution was clarified by passage through
a 0.2 micrometer cellulose acetate filter (Corning, N.Y.).
[0383] This clarified solution (total volume approximately 600 ml)
was loaded on to a Poros20 HQ (Perseptive Biosystems, MA) 1.times.2
cm column pre-equilibrated with Anion buffer (0.05 M Na acetate, pH
5.3, 0.1 M NaCl) at a flow rate of 10 ml/minute (800 cm/hour). The
column and the solution added were at ambient temperature
throughout this purification step. The column was subsequently
washed with 10 column volumes of Anion buffer.
[0384] Material that had inhibitory activity, detected following
the procedure below, in the factor Xa amidolytic assay was eluted
with Cation buffer containing 0.55 M NaCl at a flow rate of 5
ml/minute (400 cm/hour).
[0385] A sample of solution was tested in a factor Xa amidolytic
assay as follows. Reaction mixtures (150 microliters) were prepared
in 96-well plates containing factor Xa and various dilutions of the
sample in assay buffer (100 mM Tris-HCl pH 7.4; 140 mM NaCl; 0.1%
BSA). Human factor X was purchased from Enzyme Research
Laboratories (South Bend, Ind., USA) and activated with Russell's
Viper venom using the procedure of Bock, P. E., Craig, P. A.,
Olson, S. T., and Singh P., Arch. Biochem. Biophys., 273: 375-388
(1989). Following a 30 minute incubation at ambient temperature,
the enzymatic reactions were initiated by addition of 50
microliters of a 1 mM substrate solution in water
(N-alpha-benzyloxycarbo- nyl-D-arginyl-L-glycyl-L-arginine
p-nitroanilide-dihydrochloride; S-2765; Chromogenix, Molndal,
Sweden) to yield final concentrations of 0.2 nM factor Xa and 0.25
mM S-2765. Substrate hydrolysis was monitored by continuously
measuring absorbance at 405 nm using a Vmax kinetic plate reader
(Molecular Devices, Menlo Park, Calif., USA).
[0386] (D) Heat Treatment.
[0387] Half of the 0.55 M NaCl elution pool (3 ml) from
anion-exchange chromatography was neutralized by adding 1 M
Tris-HCl, pH 7.5 to a final concentration of 50 mM, incubated for 5
minutes at 90.degree. C. in a glass tube and subsequently cooled
rapidly on ice. Insoluble material was pelleted by centrifugation
19,000.times.g.sub.max at 4.degree. C. for 20 minutes. The
supernatant contained material which inhibited factor Xa in the
factor Xa amidolytic assay. About 89% of the factor Xa inhibitory
activity was recovered in the supernatant, after this heat
treatment after accounting for dilution.
[0388] (E) Molecular Sieve Chromatography using Superdex30
(Alternative for the Heat Treatment Step).
[0389] Half of the 0.55 M NaCl elution pool (3 ml) from
anion-exchange chromatography was loaded on a Superdex30 PG
(Pharmacia, Sweden) 1.6.times.66 cm column pre-equilibrated with
0.01M sodium phosphate, pH 7.4, 0.15 M NaCl at 24.degree. C. The
chromatography was conducted at a flow rate of 2 ml/minute. The
factor Xa inhibitory activity (determined in the factor Xa
amidolytic assay) eluted 56-64 ml into the run (K.sub.av of 0.207).
This elution volume would be expected for a globular protein with a
molecular mass of 14,000 daltons.
[0390] (F) Reverse Phase Chromatography.
[0391] Hookworm lysate which was fractionated by chromatography on
Concanavalin A Sepharose, anion-exchange and Superdex30 (or with
the alternative heat treatment step) was loaded on to a
0.46.times.25 cm C18 column (218TP54 Vydac; Hesperia, Calif.) which
was then developed with a linear gradient of 10-35% acetonitrile in
0.1% (v/v) trifluoroacetic acid at a flow rate of 1 ml/minute with
a rate of 0.625% change in acetonitrile/minute. FXa inhibitory
activity (determined in the factor Xa amidolytic assay) eluted at
approximately 30% acetonitrile. The HPLC runs were performed on a
Vista 5500 connected with a Polychrom 9600 detector set at 215 nm
(Varian, Calif.). Detector signals were integrated on a 4290
integrator obtained from the same company. Factor Xa inhibitory
activity containing fractions were vacuum dried and then
redissolved in PBS (0.01 M sodium phosphate, pH 7.4, 0.15 M
NaCl).
[0392] These fractions were pooled and then loaded on to a
0.46.times.25 cm C18 column (218TP54 Vydac; Hesperia, Calif.) which
was developed with a linear gradient of 10-35% acetonitrile in 0.1%
trifluoroacetic acid at a flow rate of 1 ml/minute with a slower
rate of 0.4% change in acetonitrile/minute. Factor Xa inhibitory
activity containing fractions were pooled and subsequently vacuum
dried.
[0393] (G) Molecular Weight Determination of NAP from Ancylostoma
caninum.
[0394] The estimated mass for NAP isolated as described in this
example was determined using electrospray ionisation mass
spectrometry.
[0395] A vacuum-dried pellet of NAP was dissolved in 50% (v/v)
acetonitrile, 1% (v/v) formic acid. Mass analysis was performed
using a VG Bio-Q (Fisons Instruments, Manchester UK).
[0396] The NAP sample was pumped through a capillary and at its tip
a high voltage of 4 kV was applied. Under the influence of the high
electric field, the sample was sprayed out in droplets containing
the protein molecules. Aided by the drying effect of a neutral gas
(N.sub.2) at 60.degree. C., the droplets were further reduced in
size until all the solvent had been evaporated and only the protein
species remained in the gaseous form. A population of protein
species arose which differed from each other in one charge. With a
quadrupole analyzer, the different Da/e (mass/charge)-values were
detected. Calibration of the instrument was accomplished using
Horse Heart Myoglobin (Sigma, Mo.).
[0397] The estimated mass of NAP isolated as described in sections
A, B, C, D, and F of this example is 8734.60 daltons. The estimated
mass of native NAP isolated as described in sections A, B, C, E,
and F is 8735.67 daltons.
[0398] (H) Amino Acid Sequencing of NAP from Ancylostoma
caninum.
[0399] Amino acid determination was performed on a 476-A
Protein/Peptide Sequencer with On Board Microgradient PTH Analyzer
and Model 610A Data Analysis System (Applied Biosystems, CA).
Quantification of the residues was performed by on-line analysis on
the system computer (Applied Biosystems, CA); residue assignment
was performed by visual analysis of the HPLC chromatograms. The
first twenty amino acids of the amino-terminus of native NAP were
determined to be:
3 Lys Ala Tyr Pro Glu Cys Gly Glu [SEQ. ID. NO. 97] Asn Glu Trp Leu
Asp Asp Cys Gly Thr Gln Lys Pro.
[0400] The cysteine residues were not directly detected in this
analysis because the sample was not reduced and subsequently
alkylated. Cysteines were assigned to the positions where no
specific amino acid was identified.
Example 2
[0401] Cloning and Sequencing of NAP from Ancylostoma caninum.
[0402] (A) Preparation Of Hybridization Probe.
[0403] Full-length cDNA clones encoding NAP were isolated by
screening a cDNA library, prepared from the mRNA isolated from the
nematode, Ancylostoma caninum, with a radiolabeled degenerate
oligonucleotide whose sequence was based on the first eleven amino
acids of the amino-terminus of NAP from A. caninum:
4 Lys Ala Tyr Pro Glu Cys Gly Glu [SEQ. ID. NO. 93] Asn Glu
Trp.
[0404] The 33-mer oligonucleotide hybridization probe, designated
YG99, had the following sequence:
5 AAR GCi TAY CCi GAR TGY GGi GAR [SEQ. ID. NO. 94] AAY GAR TGG
[0405] where "R" refers to A or G; "Y" refers to T or C; and "i"
refers to inosine. YG99 was radiolabeled by enzymatic 5'-end
phosphorylation (5'-end labeling kit; Amersham, Buckinghamshire,
England) using gamma-.sup.32P-ATP (specific activity>7000
Ci/mmole; ICN, Costa Mesa, Calif., USA) and subsequently passed
over a NAP.TM.10 column (Pharmacia, Uppsala, Sweden).
[0406] (B) Preparation of cDNA Library.
[0407] A cDNA library was constructed using described procedures
(Promega Protocols and Applications Guide 2nd Ed.; Promega Corp.,
Madison, Wis., USA).
[0408] Adult hookworms, Ancylostoma caninum, were purchased from
Antibody Systems (Bedford, Tex.). Poly(A+) RNA was prepared using
the QuickPrep mRNA Purification Kit (Pharmacia). About 3 micrograms
of mRNA were reverse transcribed using an oligo(dT)-NotI
primer/adaptor, AATTCGCGGCCGC(T).sub.15 [SEQ. ID. NO. 95], (Promega
Corp.) and AMV (Avian Myeloblastosis Virus) reverse transcriptase
(Boehringer, Mannheim, Germany). The enzymes used for
double-stranded cDNA synthesis were the following: E. coli DNA
polymerase I and RNaseH from Life Technologies (Gaithersburg, Md.,
USA) and T4 DNA polymerase from Pharmacia.
[0409] EcoRI linkers (pCGGAATTCCG) [SEQ. ID. NO. 98] were ligated
onto the obtained cDNA after treatment with EcoRI methylase
(RiboClone EcoRI Linker Ligation System; Promega).
[0410] The cDNAs were digested with NotI and EcoRI, passed over a
1.5% agarose gel (all sizeable material was eluted using the
Geneclean protocol, BIO101 Inc., La Jolla, Calif.), and
unidirectionally ligated into the EcoRI-NotI arms of the lambda
gt11 Sfi-NotI vector (Promega). After in vitro packaging
(GigapackII-Gold, Stratagene, La Jolla, Calif.) recombinant phage
were obtained by infecting strain Y1090 (Promega).
[0411] The usefulness of the cDNA library was demonstrated by PCR
analysis (Taq polymerase from Boehringer; 30 temperature cycles: 1
minute at 95.degree. C.; 1 minute at 50.degree. C.; 3 minutes at
72.degree. C.) of a number of randomly picked clones using the
lambda gt11 primer #1218, having the sequence, GGTGGCGACG
ACTCCTGGAG CCCG (New England Biolabs, Beverly, Mass., USA) [SEQ.
ID. NO. 96]; targeting sequences located upstream of the cDNA
insert) in combination with the above-mentioned oligo(dT)-NotI
primer/adaptor; the majority of the clones was found to contain
cDNA inserts of variable size.
[0412] (C) Identification of Clones.
[0413] Approximately 1.times.10.sup.6 cDNA clones (duplicate
plaque-lift filters were prepared using Hybond.TM.-N; Amersham)
were screened with the radiolabeled YG99 oligonucleotide using the
following pre-hybridization and hybridization conditions:
5.times.SSC (SSC: 150 mM NaCl, 15 mM trisodium citrate), 5.times.
Denhardt's solution, 0.5% SDS, 100 micrograms/ml sonicated fish
sperm DNA (Boehringer), overnight at 42.degree. C. The filters were
washed 4 times in 2.times.SSC, 0.1% SDS at 37.degree. C. After
exposure (about 72 hours) to X-ray film, a total of between 350 and
500 hybridization spots were identified.
[0414] Twenty-four positive clones, designated NAP1 through NAP24,
were subjected to a second hybridization round at lower
plaque-density; except for NAP24, single plaques containing a
homogeneous population of lambda phage were identified. The
retained clones were analyzed by PCR amplifications (Taq polymerase
from Boehringer; 30 temperature cycles: 1 minute at 95.degree. C.;
1 minute at 50.degree. C.; 1.5 minutes at 72.degree. C.) using the
oligo(dT)-NotI primer (AATTCGCGGC CGC(T).sub.15) [SEQ. ID. NO. 95]
in combination with either (i) YG99 or (ii) the lambda gt11 primer
#1218. The majority of the clones (20 out of 23) yielded a fragment
of about 400 bp when the oligo(dT)-NotI/YG99 primer set was used
and a fragment of about 520 bp when the oligo(dT)-NotI/#1218 primer
couple was used. Nineteen such possibly full-length clones were
further characterized.
[0415] The cDNA inserts of five clones were subcloned as SfiI-NotI
fragments on both pGEM-5Zf(-) and pGEM-9Zf(-) (Promega). Because
the SfiI sites of lambda gt11 and pGEM-5Zf(-) are not compatible
with one another, the cloning on this vector required the use of a
small adaptor fragment obtained after annealing the following two
5'-end phosphorylated oligonucleotides: pTGGCCTAGCG TCAGGAGT [SEQ.
ID. NO. 99] and pCCTGACGCTA GGCCATGG [SEQ. ID. NO. 100]. Following
preparation of single-stranded DNA, the sequences of these cDNAs
were determined with the dideoxy chain termination method using
primer #1233 having the sequence, AGCGGATAAC AATTTCACAC AGGA (New
England Biolabs) [SEQ. ID. NO. 101]. All five clones were found to
be full-length including a complete secretion signal. Clones NAP5,
NAP7 and NAP22 were found to have an identical coding region.
Clones NAP6 and NAP11 are also identical but differ from the NAP5
type of coding region. FIG. 1 depicts the nucleotide sequence of
the NAP5 gene and FIG. 2 depicts the amino acid sequence of the
protein encoded, AcaNAP5. Likewise, FIG. 3 depicts the nucleotide
sequence of the NAP6 [SEQ. ID. NO. 5] gene and FIG. 4 depicts the
amino acid sequence of the protein encoded, AcaNAP6 [SEQ. ID. NO.
6].
[0416] Fourteen other possibly full-length clones were subjected to
a restriction analysis. The above mentioned 400 bp PCR product
obtained with the YG99/oligo(dT)-NotI primer couple, was digested
with four different enzymes capable of discriminating between a
NAP5- and NAP6-type of clone: Sau96I, Sau3AI, DdeI, and HpaII. The
results were consistent with 10 out of the 14 clones being
NAP5-type (e.g. NAP4, NAP8, NAP9, NAP15, NAP16, NAP17, NAP18,
NAP20, NAP21, and NAP23) while the remaining four were NAP6-type
(e.g. NAP10, NAP12, NAP14, and NAP19).
[0417] These clones were renamed to reflect origin from Ancylostoma
caninum by placing the letters Aca immediately before the NAP
designation. For example, NAP5 became AcaNAP5, NAP6 became AcaNAP6
and so forth.
Example 3
[0418] Production and Purification of Recombinant AcaNAP5 in P.
pastoris.
[0419] (A) Expression Vector Construction.
[0420] The Pichia pastoris yeast expression system, including the
E. coli/P. pastoris shuttle vector, pHILD2, has been described in a
number of United States patents. See, e.g., U.S. Pat. Nos.
5,330,901; 5,268,273; 5,204,261; 5,166,329; 5,135,868; 5,122,465;
5,032,516;. 5,004,688; 5,002,876; 4,895,800; 4,885,242; 4,882,279;
4,879,231; 4,857,467; 4,855,231; 4,837,148; 4,818,700; 4,812,405;
4,808,537; 4,777,242; and 4,683,293.
[0421] The pYAM7SP8 vector used to direct expression and secretion
of recombinant AcaNAP5 in P. pastoris was a derivative of the
pHILD2 plasmid (Despreaux, C. W. and Manning, R. F., Gene 131:
35-41 (1993)), having the same general structure. In addition to
the transcription and recombination elements of pHILD2 required for
expression and chromosomal integration in P. pastoris (see Stroman,
D. W. et al., U.S. Pat. No. 4,855,23.1), this vector contained a
chimeric prepro leader sequence inserted downstream of the alcohol
oxidase (AOX1) promoter. The prepro leader consisted of the P.
pastoris acid phosphatase (PHO1) secretion signal fused to a
synthetic 19-amino acid pro-sequence. This pro-sequence was one of
the two 19-aa pro-sequences designed by Clements et al., Gene 106:
267-272 (1991) on the basis of the Saccharomyces cerevisiae
alpha-factor leader sequence. Engineered immediately downstream
from the prepro leader sequence was a synthetic multi-cloning site
with recognition sequences for the enzymes StuI, SacII, EcoRI,
BglII, NotI, XhoI, SpeI and BamHI to facilitate the cloning of
foreign genes. NAP as expressed from pYAM7SP8 in Pichia pastoris
was first translated as a prepro-product and subsequently processed
by the host cell to remove the pre- and pro-sequences.
[0422] The structure of this vector is shown in FIG. 12. The signal
sequence (S) has the nucleic acid sequence: ATG TTC TCT CCA ATT TTG
TCC TTG GAA ATT ATT TTA GCT TTG GCT ACT TTG CAA TCT GTC TTC GCT
[SEQ. ID. NO. 102]. The pro sequence (P) has the nucleic acid
sequence: CAG CCA GGT ATC TCC ACT ACC GTT GGT TCC GCT GCC GAG GGT
TCT TTG GAC AAG AGG [SEQ. ID. NO. 103]. The multiple cloning site
(MCS) has the nucleic acid sequence:
6 CCT ATC CGC GGA ATT CAG ATC TGA [SEQ. ID. NO. 104] ATG CGG CCG
CTC GAG ACT AGT GGA TCC.
[0423] The pGEM-9Zf(-) vector (Promega) containing the AcaNAP5 cDNA
was used to isolate by amplification ("PCR-rescue") the region
encoding the mature AcaNAP5 protein (using Vent polymerase from New
England Biolabs, Beverly, Mass.; 20 temperature cycles: 1 minute at
94.degree. C., 1 minute at 50.degree. C., and 1.5 minutes at
72.degree. C.). The following oligonucleotide primers were
used:
7 [SEQ. ID. NO. 105] YG101:
GCTCGCTCTA-GAAGCTTCAG-ACATGTATAA-TCTCATGTTG-G [SEQ. ID. NO. 89]
YG103: AAGGCATACC-CGGAGTGTGG-TG
[0424] The YG101 primer, targeting C-terminal sequences, contained
a non-annealing extension which included XbaI and HindIII
restriction sites (underlined).
[0425] Following digestion with XbaI enzyme, the amplification
product, having the expected size, was isolated from gel and
subsequently enzymatically phosphorylated (T4 polynucleotide kinase
from New England Biolabs, Beverly, Mass.). After heat-inactivation
(10 minutes at at 70.degree. C.) of the kinase, the
blunt-ended/XbaI fragment was directionally cloned into the vector
pYAM7SP8 for expression purposes. The recipient vector-fragment
from pYAM7SP8 was prepared by StuI-SpeI restriction, and purified
from agarose gel. The E. coli strain, WK6 (Zell, R. and Fritz,
H.-J., EMBO J., 6: 1809-1815 (1987)], was transformed with the
ligation mixture, and ampicillin resistant clones were
selected.
[0426] Based on restriction analysis, a plasmid clone containing an
insert of the expected size, designated pYAM7SP-NAP5, was retained
for further characterization.
[0427] Sequence determination of the clone pYAM7SP-NAP5 confirmed
the precise insertion of the mature AcaNAP5 coding region in fusion
with the prepro leader signal, as predicted by the construction
scheme, as well as the absence of unwanted mutations in the coding
region.
[0428] (B) Expression of Recombinant AcaNAP5 in P. pastoris.
[0429] The Pichia pastoris strain GTS115 (his4) has been described
in Stroman, D. W. et al., U.S. Pat. No. 4,855,231. All of the P.
pastoris manipulations were performed essentially as described in
Stroman, D. W. et al., U.S. Pat. No. 4,855,231.
[0430] About 1 microgram of pYAM7SP-NAP5 plasmid DNA was
electroporated into the strain GTS115 using a standard
electroporation protocol. The plasmid was previously linearized by
SalI digestion, which theoretically facilitates the targeting and
integration of the plasmid into the his4 chromosomal locus.
[0431] The selection of a AcaNAP5 high-expressor strain was
performed essentially as described hereinbelow. His+ transformants
were recovered on MD plates (Yeast Nitrogen Base without amino
acids (DIFCO), 13.4 g/l; Biotin, 400 micrograms/L; D-glucose, 20
g/l; agar, 15 g/l). Single colonies (n=60) originating from the
electroporation were inoculated into 100 microliters of
FM22-glycerol-PTM1 medium in wells of a 96-well plate and were
allowed to grow on a plate-agitator at 30.degree. C. for 24 hours.
One liter of FM22-glycerol-PTM1 medium contained 42.87 g
KH.sub.2PO.sub.4, 5 g (NH.sub.4)2SO.sub.4,.1 g
CaSO.sub.4.2H.sub.2O, 14.28 g K.sub.2SO.sub.4, 11.7 g
MgSO.sub.4.7H.sub.2O, 50 g glycerol sterilized as a 100 ml
solution, and 1 ml of PTM1 trace mineral mix filter-sterilized. The
FM22 part of the medium was prepared as a 900 ml solution adjusted
to pH 4.9 with KOH and sterile filtered. One liter of the PTM1 mix
contained 6 g CuSO.sub.4.5H.sub.2O, 0.8 g KI, 3 g
MnSO.sub.4.H.sub.2O, 0.2 g NaMoO.sub.4.2H.sub.2O, 0.02 g
H.sub.3BO.sub.3, 0.5 g CoCl.sub.2.6H.sub.2O, 20 g ZnCl.sub.2, 5 ml
H.sub.2SO.sub.4, 65 g FeSO.sub.4.7H.sub.2O, 0.2 g biotin.
[0432] The cells were then pelleted and resuspended in fresh
FM22-methanol-PTM1 medium (same composition as above except that
the 50 g glycerol was replaced by 0.5% (v/v) methanol in order to
induce expression of the AOX1 promoter). After an additional
incubation period of 24 hours at 30.degree. C., the supernatants of
the mini-cultures were tested for the presence of secreted AcaNAP5.
Two clones that directed a high level of synthesis and secretion of
AcaNAP5, as shown by the appearance of high factor Xa inhibitory
activity in the culture medium (as measured by the amidolytic
factor Xa assay described in Example 1), were selected. After a
second screening round, using the same procedure, but this time at
the shake-flask level, one isolated host cell was chosen and
designated P. pastoris GTS115/7SP-NAP5.
[0433] The host cell, GTS115/7SP-NAP5, was shown to have a wild
type methanol-utilisation phenotype (Mut.sup.+), which demonstrated
that the integration of the expression cassette into the chromosome
of GTS115 did not alter the functionality of the genomic AOX1
gene.
[0434] Subsequent production of recombinant AcaNAP5 material was
performed in shake flask cultures, as described in Stroman, D. W.
et al., U.S. Pat. No. 4,855,231. The recombinant product was
purified from Pichia pastoris cell supernatant as described
below.
[0435] (C) Purification of Recombinant AcaNAP5.
[0436] (1) Cation Exchange Chromatography.
[0437] Following expression, the culture supernatant from
GTS115/75SP-NAP5 (100 ml) was centrifuged at 16000 r.p.m. (about
30,000.times.g) for 20 minutes before the pH was adjusted with 1N
HCl to pH 3. The conductivity of the supernatant was decreased to
less than 10 mS/cm by adding MilliQ water. The diluted supernatant
was clarified by passage through a 0.22 micrometer cellulose
acetate filter (Corning Inc., Corning, N.Y., USA)
[0438] The total volume (approximately 500 ml) of supernatant was
loaded on a Poros20 HS (Perseptive Biosystems, MA) 1.times.2 cm
column pre-equilibrated with Cation Buffer (0.05 M sodium citrate,
pH 3) at a flow rate of 5 ml/minute (400 cm/hour). The column and
the sample were at ambient temperature throughout this purification
step. The column was subsequently washed with 50 column volumes
Cation Buffer. Material that had inhibitory activity in a factor Xa
amidolytic assay was eluted with Cation Buffer containing 1M NaCl
at a flow rate of 2 ml/minute.
[0439] (2) Molecular Sieve Chromatography using Superdex30.
[0440] The 1M NaCl elution pool containing the inhibitory material
(3 ml) from the cation-exchange column was loaded on a Superdex30
PG (Pharmacia, Sweden) 1.6.times.66 cm column pre-equilibrated with
0.01 M sodium phosphate, pH 7.4, 0.15 M NaCl at ambient
temperature. The chromatography was conducted at a flow rate of 2
ml/minute. The factor Xa inhibitory activity eluted 56-64 ml into
the run (K.sub.av of 0.207). This is the same elution volume as
determined for the native molecule (Example 1, part E).
[0441] (3) Reverse Phase Chromatography.
[0442] 1 ml of the pooled fractions from the gel filtration
chromatography was loaded on to a 0.46.times.25 cm C18 column
(218TP54 Vydac; Hesperia, Calif.) which was then developed with a
linear gradient of 10-35% acetonitrile in 0.1% (v/v)
trifluoroacetic acid at 1 ml/minute with a rate of 0.4% change in
acetonitrile/minute. Factor Xa inhibitory activity, assayed as in
Example 1, eluted around 30-35% acetonitrile and was present in
several fractions. HPLC runs were performed on the same system as
described in Example 1. Fractions from several runs on this column
containing the factor Xa inhibitory activity were pooled and vacuum
dried.
[0443] (4) Molecular Weight Determination of Recombinant
AcaNAP5
[0444] The estimated mass for the main constituent isolated as
described in sections (1) to (3) of this example were determined
using the same electrospray ionisation mass spectrometry system as
described in Example 1.
[0445] The estimated mass of recombinant AcaNAP5 was 8735.69
Daltons.
[0446] (5) Amino Acid Sequencing of Recombinant AcaNAP5.
[0447] Following purification by section (1) to (3) of this
example, the recombinant AcaNAP5 from Pichia pastoris was subjected
to amino acid sequence analysis as described in Example 1. The
first five amino acids of the amino-terminus of AcaNAP5 were
determined to be: Lys-Ala-Tyr-Pro-Glu [SEQ. ID. NO. 106]. The
sequence was identical to the native NAP protein (see Example
1).
Example 4
[0448] Production and Purification of Recombinant AcaNAP6 in P.
pastoris.
[0449] (A) Expression Vector Construction.
[0450] The expression vector, pYAM7SP-NAP6, was made in the same
manner as described for pYAM7SP-NAP5 in Example 3.
[0451] (B) Expression of Recombinant AcaNAP6 in P. pastoris.
[0452] The vector, pYAM7SP-NAP6, was used to transform the Pichia
strain GTS115 (his4) as described in Example 3.
[0453] (C) Purification of AcaNAP6.
[0454] The recombinant AcaNAP6, expressed from Pichia strain GTS115
(his4) transformed with the expression vector, pYAM7SP-NAP6, was
purified as described for recombinant AcaNAP5 in Example 3.
[0455] The estimated mass of recombinant AcaNAP6 was determined, as
described in Example 3, to be 8393.84 Daltons.
[0456] The majority of the AcaNAP6 preparation had the following
amino-terminus: Lys-Ala-Tyr-Pro-Glu [SEQ. ID. NO. 106].
Example 5
[0457] Expression of Recombinant Pro-AcaNAP5 in COS Cells
[0458] (A) Expression Vector Construction.
[0459] The pGEM-9Zf(-) vector (Promega Corporation, Madison, Wis.,
USA) into which the AcaNAP5 cDNA was subcloned, served as target
for PCR-rescue of the entire AcaNAP5 coding region, including the
native secretion signal (using Vent polymerase from New England
Biolabs, Beverly, Mass., USA; 20 temperature cycles: 1 minute at
95.degree. C., 1 minute at 50.degree. C., and 1.5 minutes at
72.degree. C.). The oligonucleotide primers used were: (1) YG101,
targeting the 3'-end of the gene encoding a NAP and having the
sequence, GCTCGCTCTA GAAGCTTCAG ACATGTATAA TCTCATGTTG G [SEQ, ID.
NO. 105], and (2) YG102, targeting the 5'-end of the gene encoding
a NAP and having the sequence, GACCAGTCTA GACAATGAAG ATGCTTTACG
CTATCG [SEQ. ID. NO. 107]. These primers contain non-annealing
extensions which include XbaI restriction sites (underlined).
[0460] Following digestion with XbaI enzyme, the amplification
product having the expected size was isolated from an agarose gel
and subsequently substituted for the about 450 basepair XbaI
stuffer fragment of the pEF-BOS vector (Mizushima, S. and Nagata,
S., Nucl. Acids Res., 18: 5322 (1990)] for expression purposes. The
recipient vector-fragment was prepared by XbaI digestion and
purified from an agarose gel.
[0461] E. coli strain WK6 (Zell, R. and Fritz, H.-J., EMBO J., 6:
1809-1815 (1987)] was transformed with the ligation mixture. Thirty
randomly picked ampicillin-resistant transformants were subjected
to PCR analysis (Taq polymerase from Life Technologies Inc.,
Gaithersburg, Md., USA; 30 cycles of amplification with the
following temperature program: 1 minute at 95.degree. C., 1 minute
at 50.degree. C., and 1 minute at 72.degree. C.). Oligonucleotide
primers used were: (i) YG103 having the sequence, AAGGCATACC
CGGAGTGTGG TG [SEQ. ID. NO. 89], and matching the amino-terminus of
the region encoding mature NAP, and (ii) YG60 having the sequence,
GTGGGAGACC TGATACTCTC AAG [SEQ. ID. NO. 108], and targeting vector
sequences downstream of the site of insertion, i.e., in the
3'-untranslated region of the pEF-BOS expression cassette. Only
clones that harbor the insert in the desired orientation can yield
a PCR fragment of predictable length (about 250 basepair). Two such
clones were further characterized by sequence determination and
were found to contain the desired XbaI insert. One of the clones,
designated pEF-BOS-NAP5, was used to transfect COS cells.
[0462] (B) Transfection of COS Cells.
[0463] COS-7 cells (ATCC CRL 1651) were transfected with
pEF-BOS-NAP5, pEF-BOS containing an irrelevant insert or with
omission of DNA (mock transfections) using DEAE-dextran. The
following media and stock solutions were used with the DEAE-dextran
method:
[0464] (1) COS-medium: DMEM; 10% FBS (incubated for 30 minutes at
56.degree. C.); 0.03% L-glutamine; penicillin (50 I.U./ml) and
streptomycin (50 micrograms/ml) (all products from Life
Technologies).
[0465] (2) MEM-HEPES: MEM medium from Life Technologies Inc.,
reconstituted according to the manufacturer's specifications;
containing a 25 mM final concentration of HEPES; adjusted to pH 7.1
before filtration (0.22 micrometer).
[0466] (3) DNA solution: 6 micrograms DNA per 3 ml MEM-HEPES
[0467] (4) DEAE-dextran solution: 30 microliters DEAE-dextran stock
(Pharmacia, Uppsala, Sweden; 100 mg/ml in H.sub.2O) per 3 ml
MEM-HEPES.
[0468] (5) Transfection mixture: 3 ml of the DEAE-dextran solution
is added to 3 ml of the DNA solution and the mixture is left to
stand for 30 minutes at ambient temperature.
[0469] (6) Chloroquine solution: a 1:100 dilution of chloroquine
stock (Sigma, St. Louis, Mo., USA; 10 mM in water; filtered through
a 0.22 micrometer membrane) in COS medium.
[0470] Transient transfection of the COS cells was performed as
follows. COS cells (about 3.5.times.10.sup.6), cultured in a 175
cm.sup.2 Nunc TC-flask (Life Technologies Inc.) were washed once
with MEM-HEPES. Six ml of the transfection mixture were pipetted
onto the washed cells. After incubation for 30 minutes at ambient
temperature, 48 ml of the chloroquine solution were added and the
cells were incubated for another 4 hours at 37.degree. C. The cells
were washed one time with fresh COS-medium and finally incubated in
50 ml of the same medium at 37.degree. C.
[0471] (C) Culturing of Transfected COS Cells.
[0472] Three, four, and five days after transfection a sample of
the culture supernatants was tested in a factor Xa amidolytic assay
according to the procedure in Example 1. The results clearly
demonstrated that factor Xa inhibitory activity was accumulating in
the culture supernatant of the cells transfected with
pEF-BOS-NAP5.
[0473] The COS culture supernatant was harvested five days after
transfection and the NAP protein purified as described in Example
6.
Example 6
[0474] Purification of Recombinant Pro-AcaNAP5.
[0475] (A) Anion Exchange Chromatography.
[0476] The COS culture supernatant containing Pro-AcaNAP5 was
centrifuged at 1500 r.p.m. (about 500.times.g) for 10 minutes
before adding solid sodium acetate to a final concentration of 50
mM. The following protease inhibitors were added (all protease
inhibitors from ICN Biomedicals Inc, Costa Mesa, Calif., USA):
1.0.times.10.sup.-5 M pepstatin A
(isovaleryl-Val-Val-4-amino-3-hydroxy-6-methyl-heptanoyl-Ala-4-amino-3-hy-
droxy-6-methylheptanoic acid), 1.0.times.10.sup.-5 M leupeptin,
5.times.10.sup.-5 M AEBSF (4-(2-aminoethyl)-benzenesulfonyl
fluoride). The pH was adjusted with HCl to pH 5.3. The supernatant
was clarified by passage through a 0.2 micrometer cellulose acetate
filter (Corning Inc., Corning, N.Y., USA).
[0477] The clarified supernatant (total volume approximately 300
ml) was loaded on a Poros20 HQ (Perseptive Biosystems, MA)
1.times.2 cm column pre-equilibrated with Anion buffer (0.05 M
sodium acetate, pH 5.3, 0.1 M NaCl) at a flow rate of 10 ml/minute
(800 cm/hour). The column and the sample were at ambient
temperature throughout this purification step. The column was
subsequently washed with at least 10 column volumes of Anion
buffer. Material that had inhibitory activity in a factor Xa
amidolytic assay was eluted with Anion buffer containing 0.55 M
NaCl at a flow rate of 5 ml/minute (400 cm/hour) and was
collected.
[0478] (B) Molecular Sieve Chromatography using Superdex30.
[0479] The 0.55 M NaCl elution pool (3 ml) from the anion-exchange
chromatography was loaded on a Superdex30 PG (Pharmacia, Sweden)
1.6.times.66 cm column pre-equilibrated with 0.01 M sodium
phosphate, pH 7.4, 0.15 M NaCl at 24.degree. C. The chromatography
was conducted at a flow rate of 2 ml/minute. Material which was
inhibitory in the Factor Xa amidolytic assay eluted 56-64 ml into
the run (K.sub.av of 0.207). This was exactly the same elution
volume as determined for the native molecule.
[0480] (C) Heat Treatment.
[0481] The total pool of fractions having factor Xa inhibitory
activity was incubated for 5 minutes at 90.degree. C. in a glass
tube and subsequently cooled rapidly on ice. Insoluble material was
pelleted by centrifugation 19,000.times.g.sub.max at 4.degree. C.
for 20 minutes. The supernatant contained all of the factor Xa
inhibitory activity.
[0482] (D) Reverse Phase HPLC Chromatography.
[0483] The supernatant of the heat-treated sample was loaded onto a
0.46.times.25 cm C18 column (218TP54 Vydac; Hesperia, Calif.) which
was then developed with a linear gradient of 10-35% acetonitrile in
0.1% (v/v) trifluoroacetic acid at 1 ml/minute with a rate of 0.4%
change in acetonitrile/minute. Factor Xa inhibitory activity eluted
at approximately 30% acetonitrile. The HPLC runs were performed on
the same system as described in Example 1. Factor Xa inhibitory
activity-containing fractions were vacuum dried.
[0484] (E) Molecular Weight Determination.
[0485] The estimated mass for recombinant Pro-AcaNAP5, isolated as
described in sections A-D of this example, was determined using the
same electrospray ionisation mass spectrometry system as described
in Example 1.
[0486] The estimated mass of recombinant Pro-AcaNAP5 was 9248.4
daltons.
[0487] (F) Amino Acid Sequencing.
[0488] Following purification, the recombinant Pro-AcaNAP5 from COS
cells was subjected to amino acid analysis to determine its
amino-terminus sequence, as described in Example 1. The first nine
amino acids of the amino-terminus of Pro-AcaNAP5 was determined to
be: Arg Thr Val Arg Lys Ala Tyr Pro Glu [SEQ. ID. NO. 109].
Compared to the native AcaNAP5 protein (see Example 1), Pro-AcaNAP5
possesses four additional amino acids on its N-terminus. The amino
acid sequence of Pro-AcaNAP5 is shown in FIG. 5.
Example 7
[0489] Expression of Recombinant Pro-AcaNAP6 in COS Cells
[0490] Pro-AcaNAP6 was transiently produced in COS cells
essentially as described for Pro-AcaNAP5 in Example 5.
[0491] The AcaNAP6 coding region, including the secretion signal,
was PCR-rescued with the same two oligonucleotide primers used for
AcaNAP5: (1) YG101 targeting the 3'-end of the gene and having the
sequence, GCTCGCTCTA GAAGCTTCAG ACATGTATAA TCTCATGTTG G [SEQ. ID.
NO. 105], and (2) YG102 targeting the 5'-end of the gene and having
the sequence, GACCAGTCTA GACAATGAAG ATGCTTTACG CTATCG [SEQ. ID. NO.
107]. The YG101-primer contains a non-matching nucleotide when used
with AcaNAP6 as target (underlined T-residue; compare with FIG. 1
and FIG. 3); this mismatch results in the replacement an ATT
Ile-codon by an ATA Ile-codon. The mismatch did not markedly
influence the amplification efficiency.
[0492] The following modification from Example 5 was introduced:
twenty-four hours after transfection of the COS cells (which is
described in Example 5, section B) the COS-medium containing 10%
FBS was replaced with 50 ml of a medium consisting of a 1:1 mixture
of DMEM and Nutrient Mixture Ham's F-12 (Life Technologies). The
cells then were further incubated at 37.degree. C. and the
production of factor Xa inhibitory activity detected as described
in Example 5.
Example 8
[0493] Purification of Recombinant Pro-AcaNAP6.
[0494] (A) Anion Exchange Chromatography.
[0495] The COS culture supernatant containing Pro-AcaNAP6 was
centrifuged at 1500 r.p.m. for 10 minutes before adding solid
sodium acetate to a final concentration of 50 mM. The following
protease inhibitors were added (all protease inhibitors from ICN
Biomedicals Inc, Costa Mesa, Calif., USA): 1.0.times.10.sup.-5 M
pepstatin A (isovaleryl-Val-Val-4-ami-
no-3-hydroxy-6-methyl-heptanoyl-Ala-4-amino-3-hydroxy-6-methylheptanoic
acid), 1.0.times.10.sup.-5 M leupeptin, 5.times.10.sup.-5 M AEBSF
(4-(2-aminoethyl)-benzenesulfonyl fluoride). The pH was adjusted
with HCl to pH 5.3. The supernatant was clarified by passage
through a 0.2 micrometer cellulose acetate filter (Corning Inc.,
Corning, N.Y., USA).
[0496] The clarified supernatant (total volume approximately 450
ml) was loaded on a Poros20 HQ (Perseptive Biosystems, Mass.)
1.times.2 cm column pre-equilibrated with Anion buffer (0.05 M Na
sodium acetate, pH 5.3, 0.1 M NaCl) at a flow rate of 10 ml/minute
(800 cm/hour). The column and the sample were at ambient
temperature throughout this purification step. The column was
subsequently washed with at least 10 column volumes of Anion
buffer. Material that had inhibitory activity in a factor Xa
amidolytic assay was eluted with Anion buffer containing 0.55 M
NaCl at a flow rate of 5 ml/minute (400 cm/hour) and was
collected.
[0497] (B) Molecular Sieve Chromatography using Superdex30.
[0498] The 0.55 M NaCl elution pool (3 ml) from the anion-exchange
chromatography was loaded on a Superdex30 PG (Pharmacia, Sweden)
1.6.times.66 cm column pre-equilibrated with 0.01 M sodium
phosphate, pH 7.4, 0.15 M NaCl at 24.degree. C. The chromatography
was conducted at a flow rate of 2 ml/minute. Material which was
inhibitory in the Factor Xa amidolytic assay eluted 56-64 ml into
the run (K.sub.av of 0.207). This was exactly the same elution
volume as determined for the native NAP.
[0499] (C) Reverse Phase HPLC Chromatography.
[0500] The pooled fractions from the gel filtration were loaded
onto a 0.46.times.25 cm C18 column (218TP54 Vydac; Hesperia,
Calif.) which then was developed with a linear gradient of 10-35%
acetonitrile in 0.1% (v/v) trifluoroacetic acid at a flow rate of 1
ml/minute with a rate of 0.4% change in acetonitrile/minute. Factor
Xa inhibitory activity (assayed according to Example 1) eluted at
approximately 30% acetonitrile. The HPLC runs were performed on the
same system as described in Example 1. Factor Xa inhibitory
activity containing-fractions were vacuum dried.
[0501] (D) Molecular Weight Determination.
[0502] The estimated mass for recombinant Pro-AcaNAP6, isolated as
described in sections A to C of this example, was determined using
the same electrospray ionisation mass spectrometry system as
described in Example 1.
[0503] The estimated mass of recombinant Pro-AcaNAP6 was 8906.9
daltons.
[0504] (E) Amino Acid Sequencing.
[0505] Following purification, the recombinant Pro-AcaNAP6 from COS
cells was subjected to amino acid sequence analysis as described in
Example 1. The first five amino acids of the N-terminus of
Pro-AcaNAP6 were determined to be: Arg Thr Val Arg Lys [SEQ. ID.
NO. 110]. Compared to the native NAP protein (see Example 1),
Pro-AcaNAP6 possesses four additional amino acids on its
amino-terminus. The amino acid sequence of Pro-AcaNAP6 is shown in
FIG. 6 [SEQ. ID. NO. 8].
Example 9
[0506] The Use of NAP DNA Sequences to Isolate Genes Encoding Other
NAP Proteins.
[0507] The AcaNAP5 and AcaNAP6 cDNA sequences (from Example 2) were
used to isolate related molecules from other parasitic species by
cross-hybridization.
[0508] The pGEM-9Zf(-) vectors (Promega) containing the AcaNAP5 and
AcaNAP6 cDNAs were used to PCR-rescue the regions encoding the
mature NAP proteins (Taq polymerase from Life Technologies; 20
temperature cycles: 1 minute at 95.degree. C., 1 minute at
50.degree. C., and 1.5 minutes at 72.degree. C.). The
oligonucleotide primers used were: (1) YG109, targeting the
C-terminal sequences of cDNA encoding NAP, and having the sequence,
TCAGACATGT-ATAATCTCAT-GTTGG [SEQ. ID. NO. 88], and (2) YG103 having
the sequence, AAGGCATACC-CGGAGTGTGG-TG [SEQ. ID. NO. 89]. The YG109
primer contains a single nucleotide mismatch (underlined T-residue;
compare with the sequences shown in FIGS. 1 and 3) when used with
AcaNAP6 as target. This did not markedly influence the
amplification efficiency. The correctly sized PCR products (about
230 basepairs) were both isolated from a 1.5% agarose gel. An
equimolar mixture was radiolabeled by random primer extension (T7
QuickPrime kit; Pharmacia) and subsequently passed over a Bio-Spin
30 column (Bio-Rad, Richmond, Calif., USA).
[0509] Ancylostoma ceylanicum (Ace), Ancylostoma duodenale (Adu),
and Heligmosomoides polygyrus (Hpo) cDNA libraries were prepared
essentially as described for Ancylostoma caninum in Example 2.
[0510] Ancylostoma ceylanicum and Heligmosomoides polygyrus were
purchased from Dr. D. I. Pritchard, Department of Life Science,
University of Nottingham, Nottingham, UK. Ancylostoma duodenale was
purchased from Dr. G. A. Schad, The School of Veterinary Medicine,
Department of Pathobiology, University of Pennsylvania,
Philadelphia, Pa., USA.
[0511] In each case, the cDNAs were directionally cloned as
EcoRI-NotI fragments in lambda gt11. Approximately 2.times.10.sup.5
cDNA clones from each library (duplicate plaque-lift filters were
prepared using Hybond.TM.-N; Amersham) were screened with the
radiolabeled AcaNAP5 and AcaNAP6 fragments using the following
prehybridization and hybridization conditions: 5.times.SSC (SSC:
150 mM NaCl, 15 mM trisodium citrate), 5.times. Denhardt's
solution, 0.5% SDS, 20% formamide, 100 micrograms/ml sonicated fish
sperm DNA (Boehringer), overnight at 42.degree. C. The filters were
washed 4 times for 30 minutes in 2.times.SSC, 0.1% SDS at
37.degree. C. After exposure (about 60 hours) to X-ray film, a
total of between 100 and 200 hybridization spots were identified in
the case of Ace and Adu. A small number of very faint spots were
visible in the case of the Hpo cDNA library. For each of the
libraries, eight positives were subjected to a second hybridization
round at lower plaque-density so as to isolate single plaques.
[0512] The retained clones were further characterized by PCR
amplification of the cDNA-inserts using the oligo(dT)-NotI primer
(Promega; this is the same primer used to prepare first strand
cDNA; see Example 2) [SEQ. ID. NO. 95] in combination with the
lambda-gt11 primer #1218 having the sequence, GGTGGCGACG ACTCCTGGAG
CCCG [SEQ. ID. NO. 96] (New England Biolabs; primer #1218 targets
lambda sequences located upstream of the site of cDNA insertion).
PCR amplifications were performed as follows: Taq polymerase from
Boehringer; 30 temperature cycles: 1 minute at 95.degree. C.; 1
minute at 50.degree. C.; 1.5 minutes at 72.degree. C.
Gel-electrophoretic analysis of the PCR products clearly
demonstrated that cDNAs of roughly the same size as the AcaNAP5
cDNA (e.g., 400 to 500 bp) were obtained for each species. In
addition to these AcaNAP5-sized cDNAs, some Ace and Adu cDNAs were
estimated to be about 700 bp long.
[0513] A number of clones, containing either a 500 bp or an 800 bp
insert, were chosen for sequence determination. To that end the
cDNA inserts were subcloned, as SfiI-NotI fragments, into pGEM-type
phagemids (Promega; refer to Example 2 for details) which permit
the preparation of single stranded DNA. The sequencing results led
to the identification of six different new NAP-like proteins,
designated as follows: AceNAP4, AceNAP5, AceNAP7, AduNAP4, AduNAP7,
and HpoNAP5. The nucleotide sequences of the cDNAs as well as the
deduced amino acid sequences of the encoded proteins are shown in
FIG. 7A (AceNAP4 [SEQ. ID. NO. 9]), FIG. 7B (AceNAP5) [SEQ. ID. NO.
10], FIG. 7C (AceNAP7) [SEQ. ID. NO. 11], FIG. 7D (AduNAP4) [SEQ.
ID. NO. 12], FIG. 7E (AduNAP7) [SEQ. ID. NO. 13], and FIG. 7F
(HpoNAP5) [SEQ. ID. NO. 14]. The AceNAP4 [SEQ. ID. NO. 9) and
AduNAP7 [SEQ. ID. NO. 13] cDNAs, each about 700 bp long, each
encoded proteins which incorporated two NAP domains; the other
cDNAs isolated coded for a protein having a single NAP domain. The
AduNAP4 cDNA clone [SEQ. ID. NO. 12] was not full-length, i.e,. the
clone lacked the 5'-terminal part of the coding region; the correct
reading frame could, however, be assigned based on amino acid
sequence homology with the NAP family of related molecules.
[0514] The identified cDNA sequences can be used to produce the
encoded proteins as disclosed in Examples 3, 4, 5, and 7 using the
same or alternative suitable expression systems. Conditioned media
or cell lysates, depending on the system used, can be tested as
such or after fractionation (using such methodology as outlined in
Example 3, 4, 6 and 8) for protease inhibitory and anticoagulant
activity. Proteins that are encoded by cDNAs which hybridize to
probes derived from fragments of the AcaNAP5 gene (FIG. 1) [SEQ.
ID. NO. 3] and/or the AcaNAP6 gene (FIG. 3) [SEQ. ID. NO. 5] and
that possess serine protease inhibitory and/or anticoagulant
properties are considered to belong to the NAP family of related
molecules.
Example 10
[0515] Identification of NAP by Functional Display of cDNA Encoded
Proteins.
[0516] (A) The pDONG Series of Vectors.
[0517] The nucleotide sequences of the pDONG vectors, pDONG61 [SEQ.
ID. NO. 15], pDONG62 [SEQ. ID. NO. 16] and pDONG63 [SEQ. ID. NO.
17], derivatives of pUC119 [Vieira, J. and Messing, J., Methods in
Enzymology, 153:3-11 (1987)], are depicted in FIGS. 8A to 8C
respectively.
[0518] To construct these three vectors, HindIII and SfiI
restriction sites were added at the 5'-end and 3'-end of the
filamentous phage gene 6 by PCR amplification of the M13K07 single
stranded DNA [Vieira, J. and Messing, J., Ibid] with the G6BACKHIND
backward primer and G6FORSFI61, G6FORSFI62 or G6FORSFI63 as forward
primers. In a second PCR, the three obtained fragments were
re-amplified with G6BACKHIND and G6FORNOTBAMH as forward primer to
append NotI and BamHI sites at the 3'-end of the fragments. The
sequences of the above mentioned PCR-primers are as follows
(restriction sites are underlined):
8 [SEQ. ID. NO. 111] G6BACKHIND: ATCCGAAGCT TTGCTAACAT ACTGCGTAAT
AAG (SEQ. ID. NO. 112) G6FORSFI61: TATGGGATGG CCGACTTGGC CTCCGCCTGA
GCCTCCACCT TTATCCCAAT CCAAATAAGA [SEQ. ID. NO. 113] G6FORSFI62:
ATGGGATGGC CGACTTGGCC CTCCGCCTGA GCCTCCACCT TTATCCCAAT CCAAATAAGA
[SEQ. ID. NO. 114] G6FORSFI63: TATGGGATGG CCGACTTGGC CGATCCGCCT
GAGCCTCCAC CTTTATCCCA ATCCAAATAA [SEQ. ID. NO. 115] GAG6FORNOTBAMH:
AGGAGGGGAT CCGCGGCCGC GTGATATGGG ATGGCCGACT TGGCC
[0519] Finally, the PCR products were gel-purified, individually
digested with HindIII and BamHI and inserted between the
corresponding sites of pUC119. Sequence determination confirmed
that pDONG61, pDONG62, and pDONG63 all contained the intended
insert.
[0520] The pDONG series of vectors permit the cloning of cDNAs, as
SfiI-NotI fragments. This cloning fuses the cDNAs in each of the
three reading (translation) frames to the 3'-end of filamentous
phage gene 6 which encodes one of the phage's coat proteins.
Infection of a male-specific E. coli strain harboring a
pDONG-derivative, with VCSM13 helper phage (Stratagene, La Jolla,
Calif.), results in the rescuing of pseudo-virions which
encapsidate one specific single strand of the pDONG-derivative and
which may also incorporate a recombinant protein 6 (p6) fusion
protein in their coat. cDNAs which are such that the encoded
protein is functionally displayed on the phage surface as a
recombinant p6 fusion protein become identifiable by means of a
panning experiment described below.
[0521] (B) Transfer of the Ancylostoma caninum cDNA Library from
Lambda gt11 to the pDONG Series of Vectors.
[0522] A phage lambda preparation of the pooled A. caninum cDNA
clones (about 1.times.10.sup.6 plaques, see Example 2) was used to
PCR-rescue the cDNA inserts (Taq polymerase from Life Technologies,
Gaithersburg, Md., USA; 20 temperature cycles: 1 minute at
95.degree. C., 1 minute at 50.degree. C., and 3 minutes at
72.degree. C. followed by 10 minutes at 65.degree. C.), with the
lambda gt11 primer #1218 having the sequence, GGTGGCGACG ACTCCTGGAG
CCCG [SEQ. ID. NO. 96] (New England Biolabs, Beverly, Mass., USA;
targeting sequences located upstream of the cDNA insert) in
combination with the oligo(dT)-NotI primer/adaptor (Promega) used
for first strand cDNA synthesis. Following digestion with the
restriction enzymes SfiI and NotI, the whole size-range of
amplification products were recovered from agarose gel.
[0523] All fragments were directionally cloned into the pDONG61,
pDONG62, and pDONG63 vectors. The recipient vector-fragments were
prepared by digestion of the CsCl purified vectors with SfiI and
NotI and purification with the "Wizard.TM. PCR Preps DNA
Purification System" (Promega Corp, Madison, Wis., USA).
[0524] E. coli strain TG1 [Sambrook, J., Fritsch, E. F. and
Maniatis, T., Molecular Cloning, A Laboratory Manual, Second
Edition, volumes 1 to 3, Cold Spring Harbor Laboratory Press
(1989)] was transformed by electroporation with the pDONG/cDNA
ligation mixtures. Electrotransformed cells were incubated 1 hour
at 37.degree. C. in SOC medium [Sambrook, J. et al., Ibid.] and
plated on LB-agar containing 0.1% glucose and 100 micrograms/ml
carbenicillin (245.times.245.times.25 mm plates; Nunc).
2.2.times.10.sup.6, 1.6.times.10.sup.6, and 1.4.times.10.sup.6
carbenicillin resistant transformants were obtained with pDONG61,
pDONG62, and pDONG63, respectively. From each respective library,
designated 20L, 21L and 22L, a number of randomly picked
transformants were subjected to PCR analysis (Taq polymerase from
Life Technologies; 30 cycles of amplification with the following
temperature program: 1 minute at 95.degree. C., 1 minute at
50.degree. C., and 1 to 3 minutes at 72.degree. C.) using two
primers that match with sequences flanking the multiple cloning
site of pUC119 (primers #1224 having the sequence, CGCCAGGGTT
TTCCCAGTCA CGAC [SEQ. ID. NO. 116], and #1233 having the sequence,
AGCGGATAAC AATTTCACAC AGGA [SEQ. ID. NO. 101]; New England
Biolabs). The results showed that the vast majority of the clones
contained a cDNA-insert of variable size.
[0525] (C) Factor Xa Based Affinity-Selection of cDNA Clones
Encoding a NAP Protein.
[0526] Phage particles from the 20L, 21L and 22L libraries were
rescued as follows: each library was scraped from the plates and
grown at 37.degree. C. in 100 ml LB medium supplemented with 1%
glucose and 100 micrograms/ml carbenicillin until the optical
absorbance at 600 nm reaches the value of 0.5. After addition of
VCSM13 helper phage (Stratagene) at a multiplicity of infection
(moi) of 20, the culture was left to stand for 30 minutes at
37.degree. C. and then slowly shaken for another 30 minutes. The
cells were pelleted by centrifugation and resuspended in 250 ml LB
medium supplemented with 100 micrograms/ml carbenicillin and 50
micrograms/ml kanamycin. These cultures were allowed to grow
overnight at 30.degree. C. under vigorous agitation. The resulting
phage particles were purified by two consecutive precipitations
with polyethylene glycol/NaCl and resuspended at 1.times.10.sup.13
virions per ml in TRIS-buffered saline (0.05M Tris, 0.15M sodium
chloride, pH 7.4) (TBS). Equal amounts of phage particles from the
20L, 21L and 22L were then mixed together.
[0527] Human factor Xa (see Example 1 for preparation) was
biotinylated with biotin-XX--NHS according to manufacturer's
instructions (Pierce). The amidolytic activity of the protease was
not affected by this modification as shown by an enzymatic assay
using the chromogenic substrate S-2765 (Chromogenix; see Example
1). Streptavidin-coated magnetic beads (Dynal; 1 mg per panning
round) were washed three times with TBS and blocked in TBS
supplemented with 2% skim milk (Difco) at ambient temperature.
After one hour, the magnetic beads were washed twice with TBS
before use.
[0528] For the first round of panning, 1.times.10.sup.13 phage from
the pooled libraries were incubated for 75 minutes at 4.degree. C.
in 200 microliters of TBS buffer supplemented with 250 nM
biotinylated factor Xa, 5 mM CaCl.sub.2 and 2% skim milk. After
this time, 1 mg blocked streptavidin-coated magnetic beads,
resuspended in 200 microliters of TBS containing 5 mM CaCl.sub.2
and 2% skim milk, was added to the phage solution and incubated for
1 hour at 4.degree. C. with gentle agitation. With a magnet
(Dynal), the magnetic beads were then rinsed ten times with 500
microliters of TBS containing 0.1% Tween-20. Bound phage were
eluted from the magnetic beads by incubating them with 500
microliters of 0.1 M glycine-HCl buffer (pH 2.0) for 10 minutes.
The supernatant was neutralized with 150 microliters 1 M Tris-HCl
buffer (pH 8.0).
[0529] For phage propagation, E. coli strain TG1 [Sambrook, J.,
Fritsch, E. F. and Maniatis, T., Molecular Cloning, A Laboratory
Manual, Second Edition, volumes 1 to 3, Cold Spring Harbor
Laboratory Press (1989)] was grown at 37.degree. C. in 10 ml LB
medium until the optical absorbance at 600 nm reached the value of
0.5. The culture was infected with 650 microliters of phage eluted
from the magnetic beads and briefly incubated at 37.degree. C. with
no shaking. After centrifugation, the infected cells were
resuspended in 2 ml LB medium and plated onto
245.times.245.times.25 mm plates filled with LB-agar containing 1%
glucose and 100 micrograms/ml carbenicillin. After overnight
incubation at 37.degree. C., the cells were scraped from the plates
and resuspended in 40 ml LB medium supplemented with 1% glucose and
100 micrograms/ml carbenicillin. A cell aliquot corresponding to 15
optical densities at 600 nm was then used to inoculate 100 ml LB
medium containing 1% glucose and 100 micrograms/ml carbenicillin.
Phage rescue for the next panning round was done as outlined
above.
[0530] For the second panning round, 6.times.10.sup.12 phage were
incubated during 90 minutes with 1 mg blocked streptavidin-coated
magnetic beads in 200 microliters of TBS containing 2.5 mM
Ca.sup.2+ and 2% skim milk (this step was introduced in the
procedure to avoid selection of streptavidin-binding clones). After
removal of the beads, the same protocol was followed as for round
1. Rounds 3, 4 and 5 were accomplished as round 2, except that the
phage input was lowered to 2.times.10.sup.12 phage.
[0531] Twenty-four individual carbenicillin resistant clones that
were isolated after five rounds of panning against biotinylated
factor Xa, were then analyzed by ELISA. Streptavidin-coated 96-well
plates (Pierce) were blocked for 1 hour with 200 microliters of TBS
containing 2% skim milk per well, then were incubated for 1 hour
with 100 microliters of 20 nM biotinylated factor Xa in TBS per
well. For each clone, about 10.sup.10 phage diluted in 100
microliters TBS containing 2% skim milk and 0.1% Tween-20 were
added to the wells. After a 2-hour incubation, the wells were
rinsed four times with 200 microliters TBS containing 0.1%
Tween-20. Bound phage were visualized by consecutively incubating
with a rabbit anti-M13 antiserum (see Example 11), an alkaline
phosphatase conjugated anti-rabbit serum (Sigma), and
p-nitrophenylphosphate as substrate (Sigma). Absorbances were taken
at 405 nm after 20 minutes. Out of the 24 clones, five bound
strongly to factor Xa. No significant non-specific binding was
observed with these phage when tested in the same ELISA with
omission of biotinylated factor Xa.
[0532] Single stranded DNA was then prepared from the five positive
clones and the inserts 3' to the gene 6 were submitted to automated
DNA sequencing using the primer #1224 having the sequence,
CGCCAGGGTT TTCCCAGTCA CGAC [SEQ. ID. NO. 116] (New England
Biolabs). All five clones were found to contain the same 470 bp
5'-truncated cDNA fused in frame to gene 6 in pDONG63. The
nucleotide sequence of this cDNA as well as the deduced amino acid
sequence are depicted in FIG. 9 [SEQ. ID. NO. 19]. The cDNA,
designated AcaNAPc2, encodes a protein, designated NAP isoform c2,
that belongs to the NAP family of related proteins.
Example 11
[0533] Preparation of Antiserum Against M13 Phage.
[0534] Antiserum against M13 phage was prepared in rabbits by
subcutaneous injections of about 10.sup.13 M13K07 phage in 500
microliters of PBS (0.01 M sodium phosphate, pH 7.4+0.15 M sodium
chloride) combined with an equal volume of adjuvant. The M13K07
phage were CsCl-purified essentially as described by Glaser-Wuttke,
G., Keppner, J., and Rasched, I., Biochim. Biophys. Acta, 985:
239-247 (1989). The initial injection was done with Complete
Freunds adjuvant on day 0, followed by subsequent injections with
Incomplete Freunds adjuvant on days 7, 14 and 35. Antiserum was
harvested on day 42.
[0535] The IgG fraction of the antiserum was enriched by passage
over a Protein A-Sepharose column using conditions well known in
the art.
Example 12
[0536] The Use of AcaNAP5 and AcaNAP6 DNA Sequences to Isolate
Additional NAP-Encoding Sequences from A. caninum.
[0537] The AcaNAP5 and AcaNAP6 cDNA sequences (from Example 2) were
used to isolate related molecules from the same parasitic species
by cross-hybridization.
[0538] The pGEM-9Zf(-) vectors (Promega, Madison, Wis.) containing
the AcaNAP5 and AcaNAP6 cDNAs were used to PCR-rescue the regions
encoding the mature NAP proteins (Taq polymerase from Life
Technologies (Gaithersburg, Md.); 20 temperature cycles: 1 minute
at 95.degree. C., 1 minute at 50.degree. C., and 1.5 minutes at
72.degree. C.). The oligonucleotide primers used were: (1) YG109,
targeting the C-terminal-encoding sequences of cDNA encoding
AcaNAP5 and AcaNAP6, and having the sequence,
TCAGACATGT-ATAATCTCAT-GTTGG [SEQ. ID. NO. 88], and (2) YG103,
targeting the N-terminal-encoding sequences of mature AcaNAP5 and
AcaNAP6, having the sequence, AAGGCATACC-CGGAGTGTGG-TG [SEQ. ID.
NO. 89]. The YG109 primer contains a single nucleotide mismatch
when used with AcaNAP6 as target (underlined T-residue; compare
with the sequence shown in FIG. 3 [SEQ. ID. NO. 5]). This mismatch
did not markedly influence the amplification efficiency. The
correctly sized PCR products (about 230 basepairs) for AcaNAP5 and
AcaNAP6 were both isolated from a 1.5% agarose gel. An equimolar
mixture was radiolabeled by random primer extension (T7 QuickPrime
kit; Pharmacia (Sweden) and subsequently passed over a Bio-Spin 30
column (Bio-Rad, Richmond, Calif., USA).
[0539] Approximately 750,000 Ancylostoma caninum (Aca)cDNA clones
(refer to Example 2 (B); duplicate plaque-lift filters were
prepared using Hybond.TM.-N; Amersham (Buckinghamshire, England)
were screened with the radiolabeled AcaNAP5 and AcaNAP6 cDNA
fragments using the following prehybridization and hybridization
conditions: 5.times.SSC (SSC: 150 mM NaCl, 15 mM trisodium
citrate), 5.times. Denhardt's solution, 0.5% SDS, 20% formamide,
100 micrograms/ml sonicated fish sperm DNA (Boehringer), overnight
at 42.degree. C. The filters were washed 4 times for 30 minutes in
2.times.SSC, 0.1% SDS at 37.degree. C. After exposure to X-ray
film, a total of about 300 positives were identified.
[0540] 48 of the 300 positives were subjected to PCR-amplification
(Taq polymerase from Boehringer Mannheim, Germany; 30 temperature
cycles: 1 minute at 95.degree. C.; 1 minute at 50.degree. C.; 1.5
minutes at 72.degree. C.) using the above mentioned YG109 primer,
specific for the C-terminus-encoding sequence of AcaNAP5 and
AcaNAP6 cDNAs, and primer #1218 which targets lambda-gt11 sequences
located upstream of the site of cDNA insertion (New England
Biolabs, Beverly, Mass.; GGTGGCGACG ACTCCTGGAG CCCG [SEQ. ID. NO.
96]). 31 out of the 48 positives yielded a PCR product of a size
similar to that expected for a AcaNAP5/6-type cDNA.
[0541] The remaining 17 positives were used as template for
amplification with primer #1218 and an AcaNAPc2 specific primer
(e.g., LJ189, targeting the AcaNAPc2 C-terminus and having the
sequence GTTTCGAGTT CCGGGATATA TAAAGTCC [SEQ. ID. NO. 117]; refer
to Example 10 and FIG. 9). None of the clones yielded a PCR
product. All 17 positives were then subjected to a second
hybridization round at lower plaque-density; single isolated clones
were identified in all cases. The 17 isolated cDNA clones were
re-analyzed by PCR using the primer couples #1218/YG109 and
#1218/LJ189. Three out of the 17 clones yielded an amplification
product with the #1218/YG109 primers.
[0542] The remaining 14 clones were further analyzed by PCR
amplification with the primers #1218 and oligo(dT)-Not (Promega,
Madison, Wis.; this is the same primer used to prepare first strand
cDNA; see Example 2). All 14 clones yielded a PCR product.
Gel-electrophoretic analysis of the PCR products indicated that
some cDNAs were considerably longer than the AcaNAP5 cDNA
insert.
[0543] Ten clones, including those having the largest cDNA inserts,
were chosen for sequence determination. To that end the cDNA
inserts were subcloned as SfiI-NotI fragments onto pGEM-type
phagemids (Promega, Madison, Wis.), as described in Example 2. The
sequencing identified eight additional NAP protein sequences,
designated as follows: AcaNAP23, AcaNAP24, AcaNAP25, AcaNAP31,
AcaNAP44, AcaNAP45, AcaNAP47, and AcaNAP48. Two additional cDNA
clones, designated AcaNAP42 and AcaNAP46, encoded proteins
identical to those encoded by AcaNAP31 [SEQ. ID. NO. 34]. The
nucleotide sequences of the cDNAs as well as the deduced amino acid
sequences of the encoded proteins are shown in FIG. 13A (AcaNAP23
[SEQ. ID. NO. 31)), FIG. 13B (AcaNAP24 [SEQ. ID. NO. 32]), FIG. 13C
(AcaNAP25 [SEQ. ID. NO. 33]), FIG. 13D (AcaNAP31 [SEQ. ID. NO.
34]), FIG. 13E (AcaNAP44 [SEQ. ID. NO. 35]), FIG. 13F (AcaNAP45
[SEQ. ID. NO. 36]), FIG. 13G (AcaNAP47 [SEQ. ID. NO. 37]), and FIG.
13H (AcaNAP48 [SEQ. ID. NO. 38]). All clones were full-length and
included a complete secretion signal. The AcaNAP45 [SEQ. ID. NO.
36] and AcaNAP47 [SEQ. ID. NO. 37) cDNAs, each encode proteins
which incorporate two NAP domains; the other cDNAs code for a
protein having a single NAP domain.
Example 13
[0544] The Use of NAP DNA Sequences to Isolate Sequences Encoding a
NAP Protein from Necator americanus
[0545] The sequences of AcaNAP5 [SEQ. ID. NO. 3], AcaNAP6 [SEQ. ID.
NO. 5], AcaNAPc2 [SEQ. ID. NO. 19], AcaNAP23 [SEQ. ID. NO. 31],
AcaNAP24 [SEQ. ID. NO. 32], AcaNAP25 [SEQ. ID. NO. 33], AcaNAP31
[SEQ. ID. NO. 34], AcaNAP44 [SEQ. ID. NO. 35], AcaNAP45 [SEQ. ID.
NO. 36], AcaNAP47 [SEQ. ID. NO. 37], AcaNAP48 [SEQ. ID. NO. 38],
AceNAP4 [SEQ. ID. NO. 9], AceNAP5 [SEQ. ID. NO. 10], AceNAP7 [SEQ.
ID. NO. 11], AduNAP4 [SEQ. ID. NO. 12], AduNAP7 [SEQ. ID. NO. 13],
and HpoNAP5 [SEQ. ID. NO. 14] (see FIGS. 1, 3, 7, and 13) were used
to isolate related molecules from the hematophageous parasite
Necator americanus by PCR-cloning.
[0546] Consensus amino acid sequences were generated from regions
of homology among the NAP proteins. These consensus sequences were
then used to design the following 40 degenerate PCR primers: NAP-1,
5'-AAR-CCN-TGY-GAR-MGG-AAR-TGY-3' [SEQ. ID. NO. 90] corresponding
to the amino acid sequence
NH.sub.2-Lys-Pro-Cys-Glu-(Arg/Pro/Lys)-Lys-Cys [SEQ. ID. NO. 118];
NAP-4.RC, 5'-TW-RWA-NCC-NTC-YTT-RCA-NAC-RCA-3' [SEQ. ID. NO. 91],
corresponding to the sequence NH.sub.2-Cys-(Val/Ile/Gln)-Cys-(Ly-
s/Asp/Glu/Gln)-(Asp/Glu)-Gly-(Phe/Tyr)-Tyr [SEQ. ID. NO. 119].
These primers were used pairwise to generate NAP-specific probes by
PCR using N. americanus cDNA as template.
[0547] Adult worms, N. americanus, were purchased from Dr. David
Pritchard, University of Nottingham. Poly(A+) RNA was prepared
using the QuickPrep mRNA Purification Kit (Pharmacia, Piscataway,
N.J.). One microgram of mRNA was reverse transcribed using AMV
reverse transcriptase and random hexamer primers (Amersham,
Arlington Hills, Ill.). One fiftieth of the single-stranded cDNA
reaction product was used as template for .about.400 pmole of each
of NAP-1 and NAP-4.RC, with PCR GeneAmp (Perkin Elmer, Norwalk,
Conn.) reagents, on a Perkin-Elmer DNA thermal cycler. PCR
conditions were: cycles 1-3, denaturation at 96.degree. C. for 2
minutes, annealing at 37.degree. C. for 1 minute, and elongation at
72.degree. C. for 3 minutes (ramp time between 37.degree. C. and
72.degree. C. was 2 minutes); cycles 4-5, denaturation at
94.degree. C. for 1 minute, annealing at 37.degree. C. for 1
minute, and elongation at 72.degree. C. for 2 minutes (ramp time
between 37.degree. C. and 72.degree. C. was 2 minutes); cycles
6-45, denaturation at 94.degree. C. for 1 minutes, annealing at
37.degree. C. for 1 minute, and elongation at 72.degree. C. for 2
minutes. Elongation times were incremented by 3 seconds/cycle for
cycles 6-45.
[0548] PCR amplification of N. americanus cDNA with NAP-1 and
NAP-4.RC resulted in an approximately 100 bp amplification product.
The PCR product was labeled with (a-32P]-dCTP (Amersham) using
random primer labeling (Stratagene, La Jolla, Calif.), and labeled
DNA was separated from unincorporated nucleotides using a
Chromaspin-10 column (Clonetech, Palo Alto, Calif.).
[0549] A cDNA library was constructed using the following
procedure. Double stranded cDNA was synthesized from 1 .mu.g of N.
americanus poly(A+) RNA using AMV reverse transcriptase and random
hexamer primers (Amersham, Arlington Hills, Ill.). cDNA fragments
larger than approximately 300 bp were purified on a 6%
polyacrylamide gel and ligated to EcoRI linkers (Stratagene, San
Diego, Calif.) using standard procedures. Linkered cDNA was ligated
into EcoRI-cut and dephosphorylated lambda gt10 (Stratagene, San
Diego, Calif.) and packaged using a Gigapack Gold II packaging kit
(Stratagene, San Diego, Calif.).
[0550] Prehybridization and hybridization conditions were
6.times.SSC (SSC: 150 mM NaCl, 15 mM trisodium citrate, pH 7.0),
0.02 M sodium phosphate pH 6.5, 5.times. Denhardt's solution, 100
.mu.g/ml sheared, denatured salmon sperm DNA, 0.23% dextran
sulfate. Prehybridization and hybridization were at 42.degree. C.,
and the filters were washed for 30 minutes at 45.degree. C. with
2.times.SSC after two prewashes with 2.times.SSC for 20 minutes.
The filters were exposed overnight to X-ray film with two
intensifying screens at -70.degree. C.
[0551] Approximately 400,000 recombinant phage of the random primed
N. americanus library (unamplified) were screened with the
NAP-1/NAP-4.RC PCR fragment. About eleven recombinant phage
hybridized to this probe, of which four were isolated for
nucleotide sequencing analysis. Double stranded sequencing was
effected by subcloning the EcoRI cDNA fragments contained in these
phage isolates into pBluescript II KS+ vector (Stratagene, San
Diego, Calif.). DNA was sequenced using the Sequenase version 2.0
kit (Amersham, Arlington Hills, Ill.)) and M13 oligonucleotide
primers (Stratagene, San Diego, Calif.).
[0552] The four lambda isolates contained DNA that encoded a single
79 amino acid NAP polypeptide that resembles NAP sequences from
Ancylostoma spp. and H. polygyrus. The NAP polypeptide from N.
americanus has a calculated molecular weight of 8859.6 Daltons. The
nucleotide and deduced amino acid sequences are shown in FIG.
14.
Example 14
[0553] Expression of Recombinant AceNAP4 in COS Cells
[0554] A. Expression
[0555] AceNAP4 was transiently produced in COS cells essentially as
described for Pro-AcaNAP5 in Example 5 and Pro-AcaNAP6 in Example
7.
[0556] A pGEM-type phagemid that harbors the AceNAP4 cDNA (from
Example 9), served as target for PCR-rescue of the entire AceNAP4
coding region, including the secretion signal, using two
XbaI-appending oligonucleotide primers. The primers used were: (1)
SHPCR4, targeting the 5'-end of the gene and having the sequence,
GACCAGTCTA GACCACCATG GCGGTGCTTT ATTCAGTAGC AATA [SEQ. ID. NO.
120], and (2) SHPCR5, targeting the 3'-end of the gene and having
the sequence, GCTCGCTCTA GATTATCGTG AGGTTTCTGG TGCAAAAGTG [SEQ. ID.
NO. 121]. The XbaI restriction sites included in the primers are
underlined. The primers were used to amplify the AceNAP4 sequence
according to the conditions described in Example 5.
[0557] Following digestion with XbaI enzyme, the amplification
product, having the expected size, was isolated from an agarose gel
and subsequently substituted for the about 450 basepair XbaI
stuffer fragment of the pEF-BOS vector (Mizushima, S. and Nagata,
S., Nucl. Acids Res., 18: 5322 (1990)]. The protocol described in
Example 5 was followed to yield clone pEF-BOS-AceNAP4, which was
first shown to harbor the XbaI-insert in the desired orientation by
PCR using primers SHPCR4 and YG60, and subsequently confirmed by
sequence determination. This clone was used to transfect COS cells
according to the methods in Example 5.
[0558] Twenty-four hours after transfection of the COS cells (refer
to Example 5, section B) the COS-medium containing 10% FBS was
replaced with 50 ml of a medium consisting of a 1:1 mixture of DMEM
and Nutrient Mixture Ham's F-12 (Life Technologies (Gaithersburg,
Md.). The cells were then further incubated at 37.degree. C. and
the production of EGR-factor Xa dependent TF/factor VIIa inhibitory
activity detected as described in Example E.
[0559] B. Purification of AceNAP4
[0560] 1. Anion-Exchange chromatography
[0561] The COS culture supernatant from the AceNAP4-expressing
cells was centrifuged at 1500 r.p.m.(about 500.times.g) for 10
minutes before the following protease inhibitors (ICN Biomedicals
Inc., Costa Mesa, Calif.) were added (1.0.times.10.sup.-5M
pepstatinA (isovaleryl-Val-Val-4-amino-3-
-hydroxy-6-methyl-heptanoyl-Ala-4-amino-3hydroxy-6-methylheptanoic
acid), 1.0.times.10.sup.-5M AEBSF (4-(2-amonoethyl)-benzenesulfonyl
fluoride). Solid sodium acetate was added to a final concentration
of 50 mM before the pH was adjusted with 1N HCl to pH 5.3. The
supernatant was clarified by passage through a 0.22 micrometer
cellulose acetate filter (Corning Inc., Corning, N.Y., USA).
[0562] The clarified supernatant (total volume aproximaterly 450
ml) was loaded on a Poros20 HQ (Perseptive Biosystems, Mass.)
1.times.2 cm column preequilibrated with Anion Buffer (0.05M sodium
acetate 0.1M NaCl, pH 5.3) at a flow rate of 5 ml/minute. The
column and the sample were at ambient temperature throughout this
purification step. The column was subsequently washed with 10
column volumes of Anion Buffer and 10 column volumes of 50 mM
sodium acetate, 0.37M NaCl, pH5.3
[0563] Material that had EGR-FXa dependent fVIIa/TF amidolytic
inhibitory activity (see Example E) was eluted with 50 mM sodium
acetate, 1M NaCl, pH5.3 at a flow of 2 ml/minute.
[0564] 2. Reverse-Phase Chromatography
[0565] An aliqout of the pool of fractions collected after anion
exchange chromatography was loaded onto a 0.46.times.25 cm C18
column (218TP54 Vydac; Hesperia, Calif.) which was then developed
with a linear gradient of 10-35% acetonitrile in 0.1% (v/v)
trifluoroacetic acid at 1 ml/minute with a rate of 0.4% change in
acetonitrile/minute. EGR-FXa dependent TF/FVIIa amidolytic
inhibitory activity (see Example E) was monitored and fractions
containing this inhibitory activity were isolated and
vacuum-dried.
[0566] 3. Characterization of Recombinant AceNAP4
[0567] The AceNAP4 compound demonstrated SDS-PAGE mobility on a
4-20% gel, consistent with its size predicted from the sequence of
the cDNA (Coomassie stained gel of material after
RP-chromatography).
Example 15
[0568] Production and Purification of Recombinant AcaNAPc2 in P.
pastoris.
[0569] A. Expression Vector Construction.
[0570] Expression of the AcaNAPc2 gene in P. pastoris was
accomplished using the protocol detailed in Example 3 for the
expression of AcaNAP5 with the following modifications.
[0571] The pDONG63 vector containing the AcaNAPc2 cDNA, described
in Example 10, was used to isolate by amplification ("PCR-rescue")
the region encoding mature AcaNAPc2 protein (using Vent polymerase
from New England Biolabs, Beverly, Mass.; 20 temperature cycles: 1
minute at 94.degree. C., 1 minute at 50.degree. C., and 1.5 minutes
at 72.degree. C.). The following oligonucleotide primers were
used:
9 [SEQ. ID. NO. 122] LJ190: AAGCAACGA-TGCAGTGTGG-TGAG [SEQ. ID. NO.
123] LJ191: GCTCGCTCTA-GAAGCTTCAG-TTTCGA- GTTC-CGGGATATAT-
AAAGTCC
[0572] The LJ191 primer, targeting C-terminal sequences, contained
a non-annealing extension which included XbaI and HindIII
restriction sites (underlined).
[0573] Following digestion with XbaI enzyme, the amplification
product, having the expected size, was isolated from gel and
subsequently enzymatically phosphorylated (T4 polynucleotide kinase
from New England Biolabs, Beverly, Mass.). After heat-inactivation
(10 minutes at at 70.degree. C.) of the kinase, the
blunt-ended/XbaI fragment was directionally cloned into the vector
pYAM7SP8 for expression purposes. The recipient vector-fragment
from pYAM7SP8 was prepared by StuI-SpeI restriction, and purified
from agarose gel. The E. coli strain, WK6 (Zell, R. and Fritz,
H.-J., EMBO J., 6: 1809-1815 (1987)], was transformed with the
ligation mixture, and ampicillin resistant clones were
selected.
[0574] Based on restriction analysis, a plasmid clone containing an
insert of the expected size, designated pYAM7SP-NAPC2, was retained
for further characterization. Sequence determination of the clone
pYAM7SP-NAPC2 confirmed the precise insertion of the mature
AcaNAPc2 coding region in fusion with the prepro leader signal, as
predicted by the construction scheme, as well as the absence of
unwanted mutations in the coding region.
[0575] B. Expression of Recombinant AcaNAPc2 in P. pastoris.
[0576] The Pichia strain GTS115 (his4) has been described in
Stroman, D. W. et al., U.S. Pat. No. 4,855,231. All of the P.
pastoris manipulations were performed essentially as described in
Stroman, D. W. et al., U.S. Pat. No. 4,855,231.
[0577] About 1 microgram of pYAM7SP-NAPC2 plasmid DNA was
electroporated into the strain GTS115 using a standard
electroporation protocol. The plasmid was previously linearized by
SalI digestion, theoretically targeting the integration event into
the his4 chromosomal locus.
[0578] The selection of a AcaNAPc2 high-expresser strain was
performed as described in Example 3 for NAP isoform 5 (AcaNAP5)
using mini-culture screening. The mini-cultures were tested for the
presence of secreted AcaNAPc2 using the fVIIa/TF-EGR-fXa assay
(Example E) resulting in the selection of two clones. After a
second screening round, using the same procedure, but this time at
the shake-flask level, one isolated host cell was chosen and
designated P. pastoris GTS115/7SP-NAPc2.
[0579] The host cell, GTS115/7SP-NAPc2, was shown to have a wild
type methanol-utilisation phenotype (Mut.sup.+), which demonstrated
that the integration of the expression cassette into the chromosome
of GTS115 did not alter the functionality of the genomic AOX1
gene.
[0580] Subsequent production of recombinant AcaNAPc2 material was
performed in shake flask cultures, as described in Stroman, D. W.
et al., U.S. Pat. No. 4,855,231. The recombinant product was
purified from Pichia pastoris cell supernatant as described
below.
[0581] C. Purification of Recombinant AcaNAPc2
[0582] 1. Cation Exchange Chromatography
[0583] The culture supernatant (100 ml) was centrifuged at 16000
rpm (about 30,000.times.g) for 20 minutes before the pH was
adjusted with 1N HCl to pH 3. The conductivity of the supernatant
was decreased to less than 10 mS/cm by adding MilliQ water. The
diluted supernatant was clarified by passage through a 0.22
micrometer cellulose acetate filter (Corning Inc., Corning, N.Y.,
USA).
[0584] The total volume (approximately 500 ml) of the supernatant
was loaded onto a Poros20HS (Perseptive Biosystems, Mass.)
1.times.2 cm column pre-equilibrated with Cation Buffer (50 mM
sodium citrate pH 3) at a flow-rate of 5 ml/minute. The column and
the diluted fermentation supernatant were at room temperature
througout this purification step. The column was subsequently
washed with 50 column volumes Cation Buffer and 10 column volumes
Cation Buffer containing 0.1 M NaCl. Material that had inhibitory
activity in a prothrombinase assay was eluted with Cation Buffer
containing 1M NaCl at a flow rate of 2 ml/min.
[0585] 2. Molecular Sieve Chromatography using Superdex30
[0586] The 1M NaCl elution pool containing the EGR-fXa-fVIIa/TF
inhibitory material (3 ml; see Example C) from the cation-exchange
column was loaded onto a Superdex30 PG (Pharmacia; Sweden)
1.6.times.60 cm column pre-equilibrated with 0.1M sodium phosphate
pH7.4, 0.15M NaCl at ambient temperature. The chromatography was
conducted at a flow-rate of 2 ml/minute. The prothrombinase
inhibitory activity (Example C) eluted 56-64 ml into the run and
was pooled.
[0587] 3. Reverse Phase Chromatography
[0588] One ml of the pooled fractions from the gel filtration
chromatography was loaded onto a 0.46.times.25 cm C18 column
(218TP54 Vydac; Hesperia, Calif.) which was then developed with a
linear gradient 10-30% acetonitrile in 0.1% (v/v) trifluoroacetic
acid with a rate of 0.5% change in acetonitrile/minute. The major
peak which eluted around 20-25% acetonitrile, was manually
collected and displayed prothrombinase inhibitory activity.
[0589] 4. Molecular Mass Determination
[0590] The estimated mass for the main constituent isolated as
described in section (1) to (3) of this example was determined
using electrospray ionisation mass spectrometry. The estimated mass
of the recombinant AcaNAPc2 was 9640 daltons, fully in agreement
with the calculated molecular mass of this molecule derived from
the cDNA sequence.
Example 16
[0591] Expression of AcaNAP42 in P. pastoris.
[0592] The pGEM-9zf(-) vector (Promega) containing the AcaNAP42
cDNA (Example 12) was used to isolate the region encoding the
mature AcaNAP42 protein by PCR amplification (using Taq polymerase
from Perkin Elmer, Branchburg, N.J.; 25 temperature cycles: 1
minute at 94.degree. C., 1 minute at 50.degree. C., and 1 minute at
72.degree. C.). The following oligonucleotide primers were
used:
10 [SEQ. ID. NO. 124] oligo3: .sup.5'GAG ACT TTT AAA TCA CTG TGG
GAT CAG AAG.sup.3' [SEQ. ID. NO. 125] oligo2: .sup.5'TTC AGG ACT
AGT TCA TGG TGC GAA AGT AAT AAA.sup.3'
[0593] The oligo 3 primer, targeting the N-terminal sequence,
contained a non-annealing extension which includes DraI restriction
site (underlined). The oligo 2 primer, targeting the C-terminal
sequence, contained SpeI restriction site.
[0594] The NAP amplification product, having the expected
approximately 250 bp size, was digested with DraI and SpeI enzymes,
purified by extraction with phenol:chloroform:iso-amyl alcohol
(25:24:1, volume/volume) and precipitated in ethyl alcohol. The
recipient vector-fragment from pYAM7SP8 (Example 3) was prepared by
StuI-SpeI restriction, purified by extraction with
phenol:chloroform:iso-amyl alcohol (25:24:1, volume/volume) and
precipitated in ethyl alcohol. The E. coli strain, XL1-Blue
[Bullock, W. O., Fernande, J. M., and Short, J. M. Biotechniques 5:
376-379 (1987)], was transformed with the ligation mixture that
contained the above DNA fragments, and ampicillin resistant clones
were selected.
[0595] Based on restriction analysis, a plasmid clone containing an
insert of the expected size, designated pYAM7SP8-NAP42, was
retained for further characterization. Sequence determination of
the clone confirmed correct insertion of the mature coding region
in fusion with the PHO1/alpha-factor prepro leader signal, as
predicted by the construction scheme, as well as the absence of
unwanted mutations in the coding region.
[0596] About 10 micrograms of pYAM 7SP-NAP 42 plasmid were
electroporated into Pichia strain GTS115 (his4), described in
Example 3. The plasmid was previously digested by NotI enzyme,
targeting the integration event at the AOX1 chromosomal locus.
[0597] The His+ transformants were selected as described in Example
3. Single colonies (n=90) from the electroporation were grown in
wells of a 96-well plate containing 100 microliters of
glycerol-minimal medium for 24 hours on a plate-shaker at room
temperature. One liter of the glycerol-minimal medium contained
13.4 g Yeast Nitrogen Base without amino acids (DIFCO); 400
micrograms biotin; 10 ml glycerol; and 10 mM potassium phosphate
(pH 6.0).
[0598] The cells were pelleted and resuspended in fresh
methanol-minimal medium (same composition as above except that the
10 ml glycerol was replaced by 5 ml methanol) to induce the AOX1
promoter. After an additional incubation period of 24 hours with
agitation at room temperature, 10 microliters of culture
supernatants were tested by the Prothrombin Time Assay (Example B).
The presence of secreted AcaNAP42 was detected by the prolongation
of the coagulation time of human plasma.
Example 17
[0599] Expression of AcaNAPc2/Proline in P. pastoris.
[0600] To enhance stability and the expression level of AcaNAPc2, a
mutant cDNA was constructed that encoded an additional proline
residue at the C-terminus of the protein (AcaNAPc2/Proline or
"AcaNAPc2P"). The expression vector, pYAM7SP8-NAPc2/Proline, was
made in the same manner as described in Example 16. The oligo 8
primer is the N-terminal primer with DraI restriction site and the
oligo 9 primer is the C-terminal primer containing XbaI site and
the amino acid codon, TGG, to add one Proline residue to the
C-terminal of the natural form of AcaNAPc2.
11 [SEQ. ID. NO. 126] oligo 8: .sup.5'GCG TTT AAA GCA ACG ATG CAG
TGT GGT G.sup.3' [SEQ. ID. NO. 127] oligo 9: .sup.5'C GCT CTA GAA
GCT TCA TGG GTT TCG AGT TCC GGG ATA TAT AAA GTC.sup.3'
[0601] Following digestion of the amplification product
(approximately 270 bp) with DraI and XbaI, the amplification
product was purified and ligated with the vector-fragment from
pYAM7SP8 prepared by StuI-SpeI restriction. A plasmid clone
containing the AcaNAPc2/Proline insert was confirmed by DNA
sequencing and designated pYAM7SP8-NAPc2/Proline.
[0602] The vector, pYAM7SP8-NAPc2/Proline, was used to transform
strain GTS115 (his) as described in Example 16. Transformants were
selected and grown according to Example 16. The presence of
secreted AcaNAPc2/proline in the growth media was detected by the
prolongation of the coagulation time of human plasma (see Example
B).
Example 18
[0603] Alternative Methods of Purifying AcaNAP5. AcaNAPc2 and
AcaNAPc2P
[0604] (A) AcaNAp5
[0605] An alternative method of purifying AcaNAP5 from fermentation
media is as follows. Cells were removed from a fermentation of a
Pichia pastoris strain expressing AcaNAP5, and the media was
frozen. The purification protocol was initiated by thawing frozen
media overnight at 4.degree. C., then diluting it with
approximately four parts Milli Q water to lower the conductivity
below 8 mS. The pH was adjusted to 3.5, and the media was filtered
using a 0.22 .mu.m cellulose acetate filter (Corning Inc., Corning,
N.Y.).
[0606] The activity of the NAP-containing material was determined
in the prothrombin time clotting assay at the beginning of the
purification procedure and at each step in the procedure using the
protocol in Example B.
[0607] The filtered media was applied to a Pharmacia SP-Fast Flow
column, at a flow rate of 60 ml/min at ambient temperature, and the
column was washed with 10 column volumes of 50 mM
citrate/phosphate, pH 3.5. Step elution was performed with 100 mM
NaCl, 250 mM NaCl, and then 1000 mM NaCl, all in 50 mM
citrate/phosphate, pH 3.5. PT activity was detected in the 250 mM
NaCl eluate. The total eluate was dialyzed until the conductivity
was below 8 mS.
[0608] The pH of the material was adjusted to 4.5 with acetic acid,
and then applied to a sulfoethyl aspartamide column at ambient
temperature. Approximately 10 column volumes of 50 mM ammonium
acetate, pH 4.5/40% acetonitrile, were used to wash the column. The
column was eluted with 50 mM ammonium acetate, pH 4.5/40%
acetonitrile/200 mM NaCl, and the eluate was dialyzed or
diafiltered as before.
[0609] The eluate was adjusted to 0.1% TFA, applied to a Vydac C18
protein/peptide reverse phase column at ambient temperature, and
eluted using 0.1% TFA/19% acetonitrile, followed by 0.1% TFA/25%
acetonitrile, at a flow rate of 7 ml/min. NAP was detected in and
recovered from the 0.1% TFA/25% acetonitrile elution.
[0610] (B) AcaNAPc2 and AcaNAPc2P
[0611] AcaNAPc2 or AcaNAPc2P can be purified as described above
with the following protocol modifications. After thawing and
diluting the media to achieve a conductivity below 8 mS, the pH of
the AcaNAPc2-containing media was adjusted to pH 5.0 using NaOH.
The filtered media was applied to a Pharmacia Q Fast Flow column,
at a flow rate of 60 ml/min at ambient temperature, and the column
was washed with 10 column volumes of 50 mM acetic acid, pH 5.0.
Step elution was performed with 100 mM NaCl, 250 mM NaCl, and then
1000 mM NaCl, all in 50 mM acetic acid, pH 5.0. PT activity was
detected in the 250 mM NaCl eluate. The total eluate was dialyzed
until the conductivity was below 8 mS, and the protocol outlined
above was followed using sulfoethyl aspartamide and RP-HPLC
chromatography.
Example A
[0612] Factor Xa Amidolytic Assay.
[0613] The ability of NAPs of the present invention to act as
inhibitors of factor Xa catalytic activity was assessed by
determining the NAP-induced inhibition of amidolytic activity
catalyzed by the human enzyme, as represented by Ki* values.
[0614] The buffer used for all assays was HBSA (10 mM HEPES, pH
7.5, 150 mM sodium chloride, 0.1% bovine serum albumin). All
reagents were from Sigma Chemical Co. (St. Louis, Mo.), unless
otherwise indicated.
[0615] The assay was conducted by combining in appropriate wells of
a Corning microtiter plate, 50 microliters of HBSA, 50 microliters
of the test NAP compound diluted (0.025-25 nM) in HBSA (or HBSA
alone for uninhibited velocity measurement), and 50 microliters of
the Factor Xa enzyme diluted in HBSA (prepared from purified human
factor X obtained from Enzyme Research Laboratories (South Bend,
Ind.) according to the method described by Bock, P. E. et al.,
Archives of Biochem. Biophys. 273: 375 (1989). The enzyme was
diluted into HBSA prior to the assay in which the final
concentration was 0.5 nM). Following a 30 minute incubation at
ambient temperature, 50 microliters of the substrate S2765
(N-alpha-benzyloxycarbonyl-D-argininyl-L-glycyl-L-arginine-p-nitroanilide
dihydrochloride, obtained from Kabi Diagnostica (or Kabi Pharmacia
Hepar Inc., Franklin, Ohio) and made up in deionized water followed
by dilution in HBSA prior to the assay) were added to the wells
yielding a final total volume of 200 microliters and a final
concentration of 250 micromolar (about 5-times Km). The initial
velocity of chromogenic substrate hydrolysis was measured by the
change in absorbance at 405 nm using a Thermo Max.RTM. Kinetic
Microplate Reader (Molecular Devices, Palo alto, Calif.) over a 5
minute period in which less than 5% of the added substrate was
utilized.
[0616] Ratios of inhibited pre-equilibrium, steady-state velocities
containing NAP (Vi) to the uninhibited velocity of free fXa alone
(V.sub.o) were plotted against the corresponding concentrations of
NAP. These data were then directly fit to an equation for
tight-binding inhibitors (Morrison, J. F., and Walsh, C. T., Adv.
Enzymol. 61:201-300 (1988)], from which the apparent equilibrium
dissociation inhibitory constant K.sub.i* was calculated.
[0617] Table 1 below gives the Ki* values for the test compounds
AcaNAP5 [SEQ. ID. NO. 4], AcaNAP6 [SEQ. ID. NO. 6], and AcaNAPc2
[SEQ, ID. NO. 59], prepared as described in Examples 3, 4, and 15,
respectively. The data show the utility of AcaNAP5 and AcaNAP6 as
potent in vitro inhibitors of human FXa. In contrast, AcaNAPc2 did
not effectively inhibit FXa amidolytic activity indicating that it
does not affect the catalytic activity of free fXa.
12 TABLE 1 Compound Ki* (pM) AcaNAP5 43 .+-. 5 AcaNAP6 996 .+-. 65
AcaNAPc2 NI.sup.a .sup.aNI = no inhibition; a maximum of 15%
inhibition was observed up to 1 .mu.M.
Example B
[0618] Prothrombin Time (PT) and Activated Partial Thromboplastin
Time (aPTT) Assays.
[0619] The ex vivo anticoagulant effects of NAPs of the present
invention in human plasma were evaluated by measuring the
prolongation of the activated partial thromboplastin time (aPTT)
and prothrombin time (PT) over a broad concentration range of each
inhibitor.
[0620] Fresh frozen pooled normal citrated human plasma was
obtained from George King Biomedical, Overland Park, Kans.
Respective measurements of aPTT and PT were made using the
Coag-A-Mate RA4 automated coagulometer (General Diagnostics,
Organon Technica, Oklahoma City, Okla.) using the Automated aPTT
Platelin.RTM. L reagent (Organon Technica, Durham, N.C.) and
Simplastin.RTM. Excel (Organon Technica, Durham, N.C.)
respectively, as initiators of clotting according to the
manufacturer's instructions.
[0621] The assays were conducted by making a series of dilutions of
each tested NAP in rapidly thawed plasma followed by adding 200
microliters or 100 microliters of the above referenced reagents to
the wells of the assay carousel for the aPTT or PT measurements,
respectively. Alternatively, the NAPs were serially diluted into
HBSA and 10 .mu.l of each dilution were added to 100 .mu.l of
normal human plasma in the wells of the Coag-A-Mate assay carousel,
followed by addition of reagent.
[0622] Concentrations of NAP were plotted against clotting time,
and a doubling time concentration was calculated, i.e., a specified
concentration of NAP that doubled the control clotting time of
either the PT or the aPTT. The control clotting times (absence of
NAP) in the PT and APTT were 12.1 seconds and 28.5 seconds,
respectively.
[0623] Table 2 below shows the ex vivo anticoagulant effects of
AcaNAP5 [SEQ. ID. NO. 4], AcaNAP6 [SEQ. ID. NO. 6], AcaNAPc2 [SEQ.
ID. NO. 59], and AceNAP4 [SEQ. ID. NO. 62] and Pro-AcaNAP5 [SEQ.
ID. NO. 7] represented by the concentration of each that doubled
(doubling concentration) the control clotting time of normal human
plasma in the respective PT and APTT clotting assays relative to a
control assay where no such NAP was present. The data show the
utility of these compounds as potent anticoagulants of clotting
human plasma. The data also demonstrate the equivalency of native
NAP and recombinant NAP.
13 TABLE 2 Doubling Doubling Concentra- Concentration tion (nM) in
(nM) in the Compound the PT aPTT AcaNAP5.sup.a 43 .+-. 8 87 .+-. 4
AcaNAP6.sup.a 37 .+-. 3 62 .+-. 0 AcaNAPc2.sup.a 15 .+-. 1 105 .+-.
11 AceNAP4.sup.a 40 .+-. 4 115 .+-. 12 AcaNAP5.sup.b 26.9 76.2
AcaNAP5.sup.c 39.2 60.0 Pro-AcaNAP5.sup.d 21.9 31.0 .sup.aMade in
Pichia pastoris. .sup.bNative protein. .sup.cMade in Pichia
pastoris (different recombinant batch than (a)). .sup.dMade in COS
cells.
[0624] FIGS. 10A and 10B also show NAP-induced prolongation of the
PT (FIG. 10A) and aPTT (FIG. 10B) in a dose-dependent manner.
Example C
[0625] Prothrombinase Inhibition Assay
[0626] The ability of NAP of the present invention to act as an
inhibitor of the activation of prothrombin by Factor Xa that has
been assembled into a physiologic prothrombinase complex was
assessed by determining the respective inhibition constant,
Ki*.
[0627] Prothrombinase activity was measured using a coupled
amidolytic assay, where a preformed complex of human FXa, human
Factor Va (FVa), and phospholipid vesicles first activates human
prothrombin to thrombin. The amidolytic activity of the generated
thrombin is measured simultaneously using a chromogenic substrate.
Purified human FVa was obtained from Haematologic Technologies,
Inc. (Essex Junction, Vt.). Purified human prothrombin was
purchased from Celsus Laboratories, Inc. (Cincinnati, Ohio). The
chromogenic substrate Pefachrome t-PA (CH.sub.3SO.sub.2-D-hexa-
hydrotyrosine-glycyl-L-arginine-p-nitroanilide) from Pentapharm Ltd
(Basel, Switzerland) was purchased from Centerchem, Inc.
(Tarrytown, N.Y.). The substrate was reconstituted in deionized
water prior to use. Phospholipid vesicles were made, consisting of
phosphotidyl choline (67%, w/v), phosphatidyl glycerol (16%, w/v),
phosphatidyl ethanolamine (10%, w/v), and phosphatidyl serine (7%,
w/v) in the presence of detergent, as described by Ruf et al. [Ruf,
W., Miles, D. J., Rehemtulla, A., and Edgington, T. S. Methods in
Enzymology 222: 209-224 (1993)]. The phospholipids were purchased
from Avanti Polar Lipids, (Alabaster, Ala.).
[0628] The prothrombinase complex was formed in a polypropylene
test tube by combining FVa, FXa, and phospholipid vesicles (PLV) in
HBSA containing 3 mM CaCl.sub.2 for 10 min. In appropriate wells of
a microtiter plate, 50 .mu.l of the complex were combined with 50
.mu.l of NAP diluted in HBSA, or HBSA alone (for V.sub.o
(uninhibited velocity) measurement). Following an incubation of 30
min at room temperature, the triplicate reactions were initiated by
the addition of a substrate solution, containing human prothrombin
and the chromogenic substrate for thrombin, Pefachrome tPA. The
final concentration of reactants in a total volume of 150 .mu.L of
HBSA was: NAP (0.025-25 nM), FXa (250 fM), PLV (5 .mu.M),
prothrombin (250 nM), Pefachrome tPA (250 .mu.M, 5.times.Km), and
CaCl.sub.2 (3 mM).
[0629] The prothrombinase activity of fXa was measured as an
increase in the absorbance at 405 nm over 10 min (velocity),
exactly as described in Example A, under steady-state conditions.
The absorbance increase was sigmoidal over time, reflecting the
coupled reactions of the activation of prothrombin by the
FXa-containing prothrombinase complex, and the subsequent
hydrolysis of Pefachrome tPA by the generated thrombin. The data
from each well of a triplicate were combined and fit by
reiterative, linear least squares regression analysis, as a
function of absorbance versus time.sup.2, as described (Carson, S.
D. Comput. Prog. Biomed. 19: 151-157 (1985)] to determine the
initial velocity (V.sub.i) of prothrombin activation. Ratios of
inhibited steady-state initial velocities containing NAP (Vi) to
the uninhibited velocity of prothrombinase fXa alone (V.sub.o) were
plotted against the corresponding concentrations of NAP. These data
were directly fit to the equation for tight-binding inhibitors, as
in Example A above, and the apparent equilibrium dissociation
inhibitory constant K.sub.i* was calculated.
[0630] Table 3 below gives the dissociation inhibitor constant
(Ki*) of recombinant AcaNAP5 [SEQ. ID. NO. 4], AcaNAP6 [SEQ. ID.
NO. 6] and AcaNAPc2 [SEQ. ID. NO. 59] (all made in Pichia pastoris
as described) against the activation of prothrombin by human fXa
incorporated into a prothrombinase complex. These data show the
utility of these compounds as inhibitors of human FXa incorporated
into the prothrombinase complex.
14 TABLE 3 Compound Ki* (pM) AcaNAP5 144 .+-. 15 AcaNAP6 207 .+-.
40 AcaNAPc2 2385 .+-. 283
[0631] The data presented in Examples A, B, and C suggest that
AcaNAP5 and AcaNAP6 may be interacting with FXa in a similar manner
that involves directly restricting access of both the peptidyl and
macromolecular substrate (prothrombin) to the catalytic center of
the enzyme. In contrast, AcaNAPc2 appears to be interacting with
FXa in a way that only perturbs the macromolecular interactions of
this enzyme with either the substrate and/or cofactor (Factor Va),
while not directly inhibiting the catalytic turnover of the
peptidyl substrate (see Table 1).
Example D
[0632] In vitro Enzyme Assays for Activity Specificity
Determination
[0633] The ability of NAP of the present invention to act as a
selective inhibitor of FXa catalytic activity or TF/VIIa activity
was assessed by determining whether the test NAP would inhibit
other enzymes in an assay at a concentration that was 100-fold
higher than the concentration of the following related serine
proteases: thrombin, Factor Xa, Factor XIa, Factor XIIa,
kallikrein, activated protein C, plasmin, recombinant tissue
plasminogen activator (rt-PA), urokinase, chymotrypsin, and
trypsin. These assays also are used to determine the specificity of
NAPs having serine protease inhibitory activity.
[0634] (1) General Protocol for Enzyme Inhibition Assays
[0635] The buffer used for all assays was HBSA (Example A). All
substrates were reconstituted in deionized water, followed by
dilution into HBSA prior to the assay. The amidolytic assay for
determining the specificity of inhibition of serine proteases was
conducted by combining in appropriate wells of a Corning microtiter
plate, 50 .mu.l of HBSA, 50 .mu.l of NAP at a specified
concentration diluted in HBSA, or HBSA alone (uninhibited control
velocity, Vo), and 50 .mu.l of a specified enzyme (see specific
enzymes below). Following a 30 minute incubation at ambient
temperature, 50 .mu.l of substrate were added to triplicate wells.
The final concentration of reactants in a total volume of 200 .mu.l
of HBSA was: NAP (75 nM), enzyme (750 pM), and chromogenic
substrate (as indicated below). The initial velocity of chromogenic
substrate hydrolysis was measured as a change in absorbance at 405
nm over a 5 minute period, in which less than 5% of the added
substrate was hydrolyzed. The velocities of test samples,
containing NAP (Vi) were then expressed as a percent of the
uninhibited control velocity (Vo) by the following formula: Vi/Vo X
100, for each of the enzymes.
[0636] (2) Specific Enzyme Assays
[0637] (a) Thrombin Assay
[0638] Thrombin catalytic activity was determined using the
chromogenic substrate Pefachrome t-PA
(CH.sub.3SO.sub.2-D-hexahydrotyrosine-glycyl-L--
arginine-p-nitroaniline, obtained from Pentapharm Ltd., Basel,
Switzerland). The final concentration of Pefachrome t-PA was 250
.mu.M (about 5-15 times Km). Purified human alpha-thrombin was
obtained from Enzyme Research Laboratories, Inc. (South Bend,
Ind.).
[0639] (b) Factor Xa Assay
[0640] Factor Xa catalytic activity was determined using the
chromogenic substrate S-2765
(N-benzyloxycarbonyl-D-arginine-L-glycine-L-arginine-p-n-
itroaniline), obtained from Kabi Pharmacia Hepar, Inc. (Franklin,
Ohio). All substrates were reconstituted in deionized water prior
to use. The final concentration of S-2765 was 250 .mu.M (about
5-times Km). Purified human Factor X was obtained from Enzyme
Research Laboratories, Inc. (South Bend, Ind.) and Factor Xa (FXa)
was activated and prepared from Factor X as described (Bock, P. E.,
Craig, P. A., Olson, S. T., and Singh, P. Arch. Biochem. Biophys.
273:375-388 (1989)].
[0641] (c) Factor XIa Assay
[0642] Factor FXIa catalytic activity was determined using the
chromogenic substrate S-2366
(L-Pyroglutamyl-L-prolyl-L-arginine-p-nitroaniline, obtained from
Kabi Pharmacia Hepar, Franklin, Ohio). The final concentration of
S-2366 was 750 .mu.M. Purified human FXIa was obtained from Enzyme
Research Laboratories, Inc.(South Bend, Ind.).
[0643] (d) Factor XIIa Assay
[0644] Factor FXIIa catalytic activity was determined using the
chromogenic substrate Spectrozyme FXIIa
(H-D-CHT-L-glycyl-L-arginine-p-ni- troaniline), obtained from
American Diagnostica, Greenwich, Conn.). The final concentration of
Spectrozyme FXIIa was 100 .mu.M. Purified human FXIIa was obtained
from Enzyme Research Laboratories, Inc. (South Bend, Ind.).
[0645] (e) Kallikrein Assay
[0646] Kallikrein catalytic activity was determined using the
chromogenic substrate S-2302
(H-D-prolyl-L-phenylalanyl-L-arginine-p-nitroaniline, obtained from
Kabi Pharmacia Hepar, Franklin, Ohio). The final concentration of
S-2302 was 400 .mu.M. Purified human kallikrein was obtained from
Enzyme Research Laboratories, Inc. (South Bend, Ind.).
[0647] (f) Activated Protein C (aPC)
[0648] Activated Protein C catalytic activity was determined using
the chromogenic substrate Spectrozyme PCa
(H-D-lysyl(-Cbo)-L-prolyl-L-arginin- e-p-nitroaniline) obtained
from American Diagnostica Inc. (Greenwich, Conn.). The final
concentration was 400 .mu.M (about 4 times Km). Purified human aPC
was obtained from Hematologic Technologies, Inc. (Essex Junction,
Vt.)
[0649] (g) Plasmin Assay
[0650] Plasmin catalytic activity was determined using the
chromogenic substrate S-2366
(L-Pyroglutamyl-L-prolyl-L-arginine-p-nitroaniline, obtained from
Kabi Pharmacia Hepar, Franklin, Ohio). The final concentration of
S-2366 was 300 .mu.M (about 4 times Km). Purified human plasmin was
obtained from Enzyme Research Laboratories, Inc. (South Bend,
Ind.).
[0651] (h) Recombinant Tissue Plasminoaen Activator (rt-PA)
[0652] rt-PA catalytic activity was determined using the substrate,
Pefachrome t-PA
(CH.sub.3SO.sub.2-D-hexahydrotyrosine-glycyl-L-arginine-p-
-nitroaniline, obtained from Pentapharm Ltd., Basel, Switzerland).
The final concentration was 500 .mu.M (about 3 times Km). Human
rt-PA (Activase.RTM.) was obtained from Genentech, Inc. (So. San
Fransisco, Calif.).
[0653] (i) Urokinase
[0654] Urokinase catalytic activity was determined using the
substrate S-2444
(L-Pyroglutamyl-L-glycyl-L-arginine-p-nitroaniline, obtained from
Kabi Pharmacia Hepar, Franklin, Ohio). The final concentration of
S-2444 was 150 .mu.M (about 7 times Km). Human urokinase
(Abbokinase.RTM.), purified from cultured human kidney cells, was
obtained from Abbott Laboratories (North Chicago, Ill.).
[0655] (j) Chymotrypsin
[0656] Chymotrypsin catalytic activity was determined using the
chromogenic substrate, S-2586
(Methoxy-succinyl-L-argininyl-L-prolyl-L-ty- rosine-p-nitroaniline,
which was obtained from Kabi Pharmacia Hepar, Franklin, Ohio). The
final concentration of S-2586 was 100 .mu.M (about 8 times Km).
Purified (3.times.-crystallized; CDI) bovine
pancreatic-chymotrypsin was obtained from Worthington Biochemical
Corp. (Freehold, N.J.).
[0657] (k) Trypsin
[0658] Trypsin catalytic activity was determined using the
chromogenic substrate S-2222
(N-benzoyl-L-isoleucyl-L-glutamyl[-methyl
ester]-L-arginine-p-nitroaniline, which was obtained from Kabi
Pharmacia Hepar, Franklin, Ohio). The final concentration of S-2222
was 300 .mu.M (about 5 times Km). Purified human pancreatic trypsin
was obtained from Scripps Laboratories (San Diego, Calif.).
[0659] Table 4 lists the inhibition of the amidolytic acativity of
FXa and 10 additional serine proteases by either recombinant
AcaNAP-5 [SEQ. ID. NO. 4] or recombinant AcaNAP-6 [SEQ. ID. NO. 6]
(both expressed in Pichia pastoris, as described), expressed as
percent of control velocity. These NAPs demonstrate a high degree
of specificity for the inhibition of FXa compared to the other,
related serine proteases.
15 TABLE 4 % Control % Control Velocity Velocity Enzyme +AcaNAP5
+AcaNAP6 FXa 1 .+-. 1 14 .+-. 1 FIIa 104 .+-. 5 98 .+-. 3 FXIa 34
.+-. 12 98 .+-. 3 FXIIa 103 .+-. 6 100 .+-. 4 kallikrein 102 .+-. 4
101 .+-. 3 aPC 95 .+-. 2 98 .+-. 1 plasmin 111 .+-. 6 113 .+-. 12
r-tPA 96 .+-. 9 96 .+-. 7 urokinase 101 .+-. 14 96 .+-. 2
chymotrypsin 105 .+-. 0 100 .+-. 11 trypsin 98 .+-. 6 93 .+-. 4
[0660] Table 5 lists the inhibitory effect of recombinant AcaNAPc2
[SEQ. ID. NO. 59} and recombinant AceNAP4 [SEQ. ID. NO. 62] (both
expressed in Pichia pastoris, as described) on the amidolytic
activity of 11 selected serine proteases. Inhibition is expressed
as percent of control velocity. These data demonstrate that these
NAPs possess a high degree of specificity for the serine proteases
in Table 5.
16 TABLE 5 % Control % Control Velocity Velocity Enzyme +AcaNAPc2
+AceNAP4 FXa 84 .+-. 3 76 .+-. 3 FIIa 99 .+-. 3 93 .+-. 3 FXIa 103
.+-. 4 96 .+-. 1 FXIIa 97 .+-. 1 102 .+-. 2 kallikrein 101 .+-. 1
32 .+-. 1 aPC 97 .+-. 3 103 .+-. 1 plasmin 107 .+-. 9 100 .+-. 1
r-tPA 96 .+-. 2 108 .+-. 3 urokinase 97 .+-. 1 103 .+-. 4
chymotrypsin 99 .+-. 0 96 .+-. 4 trypsin 93 .+-. 4 98 .+-. 4
Example E
[0661] Assays for Measuring the Inhibition of the fVIIa/TF Complex
by NAP
[0662] (1) fVIIa/TF fIX Activation Assay
[0663] This Example measures the ability of NAPs of the present
invention to act as an inhibitor of the catalytic complex of
fVIIa/TF, which has a primary role in initiation of the coagulation
response in the ex vivo prothrombin time assay (Example B).
Activation of tritiated Factor IX by the rFVIIa/rTF/PLV complex was
assessed by determining the respective intrinsic inhibition
constant, Ki*.
[0664] Lyophilized, purified, recombinant human factor VIIa was
obtained from BiosPacific, Inc. (Emeryville, Calif.),.and
reconstituted in HBS (10 mM HEPES, pH 7.5, 150 mM sodium chloride)
prior to use. Purified human Factor X was obtained from Enzyme
Research Laboratories, Inc. (South Bend, Ind.) and Factor Xa (free
FXa) was activated and prepared from Factor X as described (Bock,
P. E., Craig, P. A., Olson, S. T., and Singh, P. Arch. Biochem.
Biophys. 273:375-388 (1989)). Active site-blocked human Factor Xa
(EGR-FXa), which had been irreversibly inactivated with
L-Glutamyl-L-glycyl-L-arginyl chloromethylketone, was obtained from
Haematologic Technologies, Inc. (Essex Junction, Vt.). Recombinant
human tissue factor (rTF) was produced by a baculovirus-expression
system, and purified to homogeneity by monoclonal antibody affinity
chromatography (Corvas International, Inc., San Diego, Calif.).
[0665] The purified rTF apoprotein was incorporated into
phospholipid vesicles (rTF/PLV), consisting of phosphotidyl choline
(75%, w/v) and phosphotidyl serine (25%, w/v) in the presence of
detergent, as described by Ruf et al. (Ruf, W., Miles, D. J.,
Rehemtulla, A., and Edgington, T. S. Methods in Enzymology 222:
209-224 (1993)). The phospholipids were purchased from Avanti Polar
Lipids, (Alabaster, Ala.). The buffer used for all assays was HBSA,
HBS containing 0.1% (w/v) bovine serum albumin. All reagents were
obtained from Sigma Chemical Co. (St. Louis, Mo.), unless otherwise
indicated.
[0666] The activation of human .sup.3H-Factor IX (FIX) by the
rFVIIa/rTF complex was monitored by measuring the release of the
radiolabelled activation peptide. Purified human fIX was obtained
from Haematologic Technologies, Inc. (Essex Junction, Vt.), and
radioactively labelled by reductive tritiation as described (Van
Lenten & Ashwell, 1971, JBC 246, 1889-1894). The resulting
tritiated preparation of FIX had a specific activity of 194
clotting units/mg as measured in immuno-depleted FIX deficient
plasma (Ortho), and retained 97% of its activity. The radiospecific
activity was 2.7.times.10.sup.8 dpm/mg. The Km for the activation
of .sup.3H-FIX by rFVIIa/rTF/PLV was 25 nM, which was equivalent to
the Km obtained for untreated (unlabelled) FIX.
[0667] The assay for Ki* determinations was conducted as follows:
rFVIIa and rTF/PLV were combined in a polypropylene test tube, and
allowed to form a complex for 10 min in HBSA, containing 5 mM
CaCl.sub.2. Aliquots of rFVIIa/rTF/PLV complex were combined in the
appropriate polypropylene microcentrifuge tubes with EGR-FXa or
free FXa, when included, and either the NAP test compound at
various concentrations, after dilution into HBSA, or HBSA alone (as
V.sub.o (uninhibited velocity) control). Following an incubation of
60 min at ambient temperature, reactions were initiated by the
addition of .sup.3H-FIX. The final concentration of the reactants
in 420 .mu.l of HBSA was: rFVIIa (50 pM], rTF [2.7 nM], PLV [ 6.4
micromolar], either EGR-FXa or free FXa (300 pM], recombinant NAP
(5-1,500 pM], .sup.3H-FIX (200 nM], and CaCl.sub.2 (5 mM]. In
addition, a background control reaction was run that included all
of the above reactants, except rFVIIa.
[0668] At specific time points (8, 16, 24, 32, and 40 min), 80
.mu.l of the reaction mixture was added to an eppendorf tube that
contained an equal volume of 50 mM EDTA in HBS with 0.5% BSA to
stop the reaction; this was followed by the addition of 160 .mu.L
of 6% (w/v) trichloroacetic acid. The protein was precipitated, and
separated from the supernatant by centrifugation at 16,000.times.g
for 6 min at 4.degree. C. The radioactivity contained in the
resulting supernatant was measured by removing triplicate aliquots
that were added to Scintiverse BD (Fisher Scientific, Fairlawn,
N.J.), and quantitated by liquid scintillation counting. The
control rate of activation was determined by linear regression
analysis of the soluble counts released over time under
steady-state conditions, where less than 5% of the tritiated FIX
was consumed. The background control (<1.0% of control velocity)
was subtracted from all samples. Ratios of inhibited steady-state
velocities (Vi), in the presence of a NAP, to the uninhibited
control velocity of rFVIIa/TF alone (V.sub.o) were plotted against
the corresponding concentrations of NAP. These data were then
directly fit to an equation for tight-binding inhibitors (Morrison,
J. F., and Walsh, C. T., Adv. Enzymol. 61:201-300 (1988)], from
which the apparent equilibrium dissociation inhibitory constant
K.sub.i* was calculated.
[0669] The data for recombinant AcaNAP5, AcaNAP6, AcaNAPc2, and
AceNAP4 (prepared as described) is presented in Table 6 following
Section B, below.
[0670] (2) Factor VIIa/Tissue Factor Amidolytic Assay
[0671] The ability of NAPs of the present invention to act as an
inhibitor of the amidolytic activity of the fVIIa/TF complex was
assessed by determining the respective inhibition constant, Ki*, in
the presence and absence of active site-blocked human Factor Xa
(EGR-fXa).
[0672] rFVIIa/rTF amidolytic activity was determined using the
chromogenic substrate S-2288
(H-D-isoleucyl-L-prolyl-L-arginine-p-nitroaniline), obtained from
Kabi Pharmacia Hepar, Inc. (Franklin, Ohio). The substrate was
reconstituted in deionized water prior to use. rFVIIa and rTF/PLV
were combined in a polypropylene test tube, and allowed to form a
complex for 10 min in HBSA, containing 3 mM CaCl.sub.2. The assay
for Ki* determinations was conducted by combining in appropriate
wells of a Corning microtiter plate 50 .mu.L of the rFVIIa/rTF/PLV
complex, 50 .mu.L of EGR-FXa, and 50 .mu.L of either the NAP test
compound at various concentrations, after dilution into HBSA, or
HBSA alone (for V.sub.o (uninhibited velocity) measurement).
Following an incubation of 30 min at ambient temperature, the
triplicate reactions were initiated by adding 50 .mu.L of S-2288.
The final concentration of reactants in a total volume of 200 .mu.L
of HBSA was: recombinant NAP (0.025-25 nM), rFVIIa (750 pM), rTF
(3.0 nM), PLV (6.4 micromolar), EGR-FXa (2.5 nM), and S-2288 (3.0
mM, 3.times.Km).
[0673] The amidolytic activity of rFVIIa/rTF/PLV was measured as a
linear increase in the absorbance at 405 nm over 10 min (velocity),
using a Thermo Max.RTM. Kinetic Microplate Reader (Molecular
Devices, Palo Alto, Calif.), under steady-state conditions, where
less than 5% of the substrate was consumed. Ratios of inhibited
pre-equilibrium, steady-state velocities (Vi), in the presence of
NAP, to the uninhibited velocity in the presence of free fXa alone
(V.sub.o) were plotted against the corresponding concentrations of
NAP. These data were then directly fit to the same equation for
tight-binding inhibitors, used in Example E.1., from which the
apparent equilibrium dissociation inhibitory constant K.sub.i* was
calculated.
[0674] Table 6 below gives the Ki* values of recombinant AcaNAPc2
[SEQ. ID. NO. 59], AceNAP4 [SEQ. ID. NO. 62], AcaNAP5 [SEQ. ID. NO.
4], and AcaNAP6 [SEQ. ID. NO. 6] (prepared in Pichia pastoris, as
described) in inhibitory assays of rFVIIa/rTF activity. The data
shows the utility of AcaNAPc2 and AceNAP4 as potent inhibitors of
the human rFVIIa/rTF/PLV complex in the absence and presence of
either free FXa or active site-blocked FXa. The in vitro activity
of AcaNAPc2P (see Example 17) was substantially the same as
AcaNAPc2.
17 TABLE 6 Ki* (pM) Amidolytic Assay .sup.3H - FIX Activation NAP
No FXa Plus EGR- No FXa +free Compound Addition FXa Addition FXa
+EGR-FXa AcaNAPc2 NI 36 .+-. 20 NI 35 .+-. 5 8.4 .+-. 1.5 AceNAP4
59,230 .+-. 378 .+-. 37 ND ND ND 3,600 AcaNAP5 NI NI NI NI NI
AcaNAP6 NI NI NI NI NI NI = no inhibition ND = not determined
Example F
[0675] In vivo Models of NAP Activity
[0676] (1) Evaluation of the Antithrombotic Activity of NAP in the
Rat Model of FeCl.sub.3-Induced Platelet-Dependent Arterial
Thrombosis
[0677] The antithrombotic (prevention of thrombus formation)
properties of NAP were evaluated using the established experimental
rat model of acute vascular thrombosis.
[0678] The rat FeCl.sub.3 model is a well characterized model of
platelet dependent, arterial thrombosis which has been used to
evaluate potential antithrombotic compounds. Kurz, K. D., Main, B.
W., and Sandusky, G. E., Thromb. Res., 60: 269-280 (1990). In this
model a platelet-rich, occlusive thrombus is formed in a segment of
the rat carotid artery treated locally with a fresh solution of
FeCl.sub.3 absorbed to a piece of filter paper. The FeCl.sub.3 is
thought to diffuse into the treated segment of artery and cause
de-endothelialization of the affected vessel surface. This results
in the exposure of blood to subendothelial structures which in turn
cause platelet adherence, thrombin formation and platelet
aggregation. The net result is occlusive thrombus formation. The
effect of a test compound on the incidence of occlusive thrombus
formation following application of FeCl.sub.3 is monitored by
ultrasonic flowtometry and is used as the primary end point. The
use of flowtometry to measure carotid artery blood flow, is a
modification of the original procedure in which thermal detection
of clot formation was employed. Kurz, K. D., Main, B. W., and
Sandusky, G. E., Thromb. Res., 60: 269-280 (1990).
[0679] (a) Intravenous Administration
[0680] Male Harlan Sprague Dawley rats (420-450 g) were acclimated
at least 72 hours prior to use and fasted for 12 hours prior to
surgery with free access to water. The animals were prepared,
anesthetized with Nembutal followed by the insertion of catheters
for blood pressure monitoring, drug and anesthesia delivery. The
left carotid artery was isolated by making a midline cervical
incision followed by blunt dissection and spreading techniques to
separate a 2 cm segment of the vessel from the carotid sheath. A
silk suture is inserted under the proximal and distal ends of the
isolated vessel to provide clearance for the placement of a
ultrasonic flow probe (Transonic) around the proximal end of the
vessel. The probe is then secured with a stationary arm.
[0681] Following surgery the animals were randomized in either a
control (saline) or treatment (recombinant AcaNAP5) group. The test
compound (prepared in P. pastoris according to Example 3) was
administered as a single intravenous bolus at the doses outlined in
Table 7 after placement of the flow probe and 5 min prior to the
thrombogenic stimulus. At t=0, a 3 mm diameter piece of filter
paper (Whatman #3) soaked with 10 .mu.L of a 35% solution of fresh
FeCl.sub.3 (made up in water) was applied to the segment of
isolated carotid artery distal to the flow probe. Blood pressure,
blood flow, heart rate, and respiration were monitored for 60
minutes. The incidence of occlusion (defined as the attainment of
zero blood flow) was recorded as the primary end point.
[0682] The efficacy of AcaNAP5 [SEQ. ID. No. 4] as an
antithrombotic agent in preventing thrombus formation in this in
vivo model was demonstrated by the dose-dependent reduction in the
incidence of thrombotic occlusion, as shown in Table 7 below.
18 TABLE 7 Treatment Dose Incidence of Group (mg/kg) n Occlusion
Saline -- 8 8/8 AcaNAP5 0.001 8 7/8 AcaNAP5 0.003 8 5/8 AcaNAP5
0.01 8 3/8* AcaNAP5 0.03 8 1/8* AcaNAP5 0.1 8 0/8* AcaNAP5 0.3 4
0/4* AcaNAP5 1.0 2 0/2* *p .ltoreq. 0.05 from saline control by
Fishers test
[0683] The effective dose which prevents 50% of thrombotic
occlusions in this model (ED.sub.50) can be determined from the
above data by plotting the incidence of occlusion versus the dose
administered. This allows a direct comparison of the antithrombotic
efficacy of AcaNAP5 with other antithrombotic agents which have
also been evaluated in this model as described above. Table 8 below
lists the ED.sub.50 values for several well known anticoagulant
agents in this model compared to AcaNAP5.
19 TABLE 8 Compound ED.sub.50.sup.a Standard Heparin 300 U/kg
Argatroban 3.8 mg/kg Hirulog .TM. 3.0 mg/kg rTAP.sup.b 0.6 mg/kg
AcaNAP5 0.0055 mg/kg .sup.aED.sub.50 is defined as the dose that
prevents the incidence of complete thrombotic occlusion in 50% of
animals tested .sup.brecombinant Tick Anticoagulant Peptide, Vlasuk
et al. Thromb. Haemostas. 70: 212-216 (1993)
[0684] (b) Subcutaneous Administration
[0685] The antithrombotic effect of AcaNAP5 compared to Low
Molecular Weight heparin (Enoxaparin; Lovenox, Rhone-Poulenc Rorer)
after subcutaneous administration was evaluated in rats using the
FeCl.sub.3 model. The model was performed in an identical manner to
that described above with the exception that the compound was
administered subcutaneously and efficacy was determined at two
different times: 30 and 150 minutes after administration. To
accomplish this, both carotid arteries were employed in a
sequential manner. The results of these experiments indicate that
AcaNAP5 [SEQ. ID. NO. 4] is an effective antithrombotic agent in
vivo after subcutaneous administration. The results are shown below
in Table 9.
20 TABLE 9 30" ED.sub.50.sup.a 150" ED.sub.50.sup.a Compound
(mg/kg) (mg/kg) Low Molecular 30.0 15.0 Weight Heparin AcaNAP5 0.07
0.015 .sup.aED.sub.50 is defined as the dose that prevents the
incidence of complete thrombotic occlusion in 50% of animals
tested.
[0686] (2) Deep Wound Bleeding Measurement
[0687] A model of deep wound bleeding was used to measure the
effect of NAP on bleeding and compare the effect with that of Low
Molecular Weight Heparin.
[0688] Male rats were anesthetized and instrumented in an identical
manner to those undergoing the FeCl.sub.3 model. However,
FeCl.sub.3 was not applied to the carotid artery. The deep surgical
wound in the neck that exposes the carotid artery was employed to
quantify blood loss over time. Blood loss was measured over a
period of 3.5 hours following subcutaneous administration of either
AcaNAP5 or LMWH. The wound was packed with surgical sponges which
were removed every 30 minutes. The sponges were subsequently
immersed in Drabkin's reagent (sigma Chemical Co., St. Louis, Mo.)
which lyses the red blood cells and reacts with hemoglobin in a
colorimetric fashion. The colorimetric samples were then quantified
by measuring absorbance at 550 nM, which provides a determination
of the amount of blood in the sponge.
[0689] The dose response characteristics for both test compounds
are shown in FIG. 15 along with efficacy data for both compounds.
AcaNAP5 [SEQ. ID. NO. 4] was much more potent than Low Molecular
Weight heparin in preventing occlusive arterial thrombus formation
in this model. Furthermore, animals treated with NAP bled less than
those treated with Low Molecular Weight heparin.
[0690] The data presented in Tables 7 and 9 and FIG. 15 clearly
demonstrate the effectiveness of NAP in preventing occlusive
thrombus formation in this experimental model. The relevance of
this data to preventing human thrombosis is clear when compared to
the other anticoagulant agents, listed in Table 8. These agents
were been evaluated in the same experimental models described
therein, in an identical manner to that described for NAPs, and in
this experimental model and have demonstrated antithrombotic
efficacy in preventing thrombus formation clinically, as described
in the following literature citations: Heparin-Hirsh, J. N. Engl.
J. Med 324:1565-1574 1992, Cairns, J. A. et al. Chest 102:
456S-481S (1992), Argatroban-Gold, H. K. et al. J. Am. Coll.
Cardiol. 21: 1039-1047 (1993); and Hirulog.TM.-Sharma, G. V. R. K.
et al. Am. J. Cardiol. 72: 1357-1360 (1993) and Lidn, R. M. et al.
Circulation 88: 1495-1501 (1993).
Example G
[0691] Pig Model of Acute Coronary Artery Thrombosis
[0692] The protocol used in these studies is a modification of a
thrombosis model which has been reported previously (Lucchesi, B.
R., et al., (1994), Brit. J. Pharmacol. 113:1333-1343).
[0693] Animals were anesthetized and instrumented with arterial and
venous catheters (left common carotid and external jugular,
respectively). A thoracotomy was made in the 4th intercostal space
and the heart was exposed. The left anterior descending (LAD)
coronary artery was isolated from the overlying connective tissue
and was instrumented with a Doppler flow probe and a 17 gauge
ligature stenosis. An anodal electrode also was implanted inside
the vessel.
[0694] Baseline measurements were taken and the NAP or placebo to
be tested was administered via the external jugular vein. Five
minutes after administration, a direct current (300 .mu.A, DC) was
applied to the stimulating electrode to initiate intimal damage to
the coronary endothelium and begin thrombus formation. Current
continued for a period of 3 hours. Animals were observed until
either 1 hour after the cessation of current or the death of the
animal, whichever came first.
[0695] Table 10 presents data demonstrating the incidence of
occlusion in animals administered AcaNAP5 or AcaNAPc2P (see Example
17) at three increasing doses of NAP. The incidence of occlusion in
the animals receiving placebo was 8/8 (100%). Time to occlusion in
placebo treated animals was 66.6.+-.7.5 min. (mean.+-.sem). Vessels
in AcaNAP treated pigs that failed to occlude during the 4 hour
period of observation were assigned an arbitrary time to occlusion
of 240 minutes in order to facilitate statistical comparisons.
[0696] The data demonstrate AcaNAP5 and AcaNAPc2P were similarly
efficacious in this setting; both prolonged the time to coronary
artery occlusion in a dose dependent manner. Furthermore, both
molecules significantly prolonged in time to occlusion at a dose
(0.03 mg/kg i.v.) that did not produce significant elevations in
bleeding. These data, and other, suggest AcaNAP5 and AcaNAPc2P have
favorable therapeutic indices.
21TABLE 10 Comparision of primary endpoints between AcaNAPc2P and
AcaNAP5 after intravenous dosing in the pig model of acute coronary
artery thrombosis. Dose Incidence of Time of Occlusion Total Blood
Loss (i.v.) Occlusion (min) (ml) (mg/kg) AcaNAP5 AcaNAPc2P AcaNAP5
AcaNAPc2P AcaNAP5 AcaNAPc2P 0.01 6/6 6/6 107 .+-. 13.0 105 .+-. 6.2
2.8 .+-. 0.8 1.6 .+-. 0.3 0.03 5/6 4/6 150 .+-. 23.2 159 .+-. 27
5.6 .+-. 1.4 4.9 .+-. 1.4 0.10 4/6 2/6.dagger. 187 .+-. 22.9* 215
.+-. 25* 43.5 .+-. 18* 17.6 .+-. 7.9* .dagger.p < 0.05 vs saline
(8/8), Fisher's Exact; *p < 0.05 vs saline, ANOVA, Dunnett's
multiple comparison test.
[0697]
Sequence CWU 1
1
* * * * *