U.S. patent application number 10/629516 was filed with the patent office on 2005-06-16 for bacterical/permability-increasing protein(bpi) deletion analogs.
Invention is credited to Burke, David, Carroll, Stephen F., Horwitz, Arnold.
Application Number | 20050130889 10/629516 |
Document ID | / |
Family ID | 22276330 |
Filed Date | 2005-06-16 |
United States Patent
Application |
20050130889 |
Kind Code |
A1 |
Horwitz, Arnold ; et
al. |
June 16, 2005 |
Bacterical/permability-increasing protein(BPI) deletion analogs
Abstract
Novel BPI deletion analogs are provided that consist of amino
acid residues 10 through 193 of mature human BPI wherein the
cysteine residue at BPI amino acid position 132 is replaced by
another amino acid. Fusion proteins comprising these analogs are
also provided, as are polynucleotides encoding these products,
materials and methods for their recombinant production,
compositions and medicaments of these products, and therapeutic
uses for these products.
Inventors: |
Horwitz, Arnold; (Los
Angeles, CA) ; Carroll, Stephen F.; (Walnut Creek,
CA) ; Burke, David; (Oakdale, CA) |
Correspondence
Address: |
MARSHALL, GERSTEIN & BORUN LLP
6300 SEARS TOWER
233 S. WACKER DRIVE
CHICAGO
IL
60606
US
|
Family ID: |
22276330 |
Appl. No.: |
10/629516 |
Filed: |
July 29, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10629516 |
Jul 29, 2003 |
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09579403 |
May 25, 2000 |
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6599880 |
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09579403 |
May 25, 2000 |
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09336402 |
Jun 18, 1999 |
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6087126 |
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09336402 |
Jun 18, 1999 |
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09099725 |
Jun 19, 1998 |
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6013631 |
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Current U.S.
Class: |
435/69.1 ;
435/212; 435/252.3; 435/320.1; 435/69.3; 514/2.2; 536/23.7 |
Current CPC
Class: |
C07K 14/4742 20130101;
A61K 38/17 20130101; A61P 31/04 20180101 |
Class at
Publication: |
514/012 ;
435/069.3; 435/320.1; 435/212; 435/252.3; 536/023.7 |
International
Class: |
A61K 038/16; C07H
021/04; C12N 015/74; C12N 009/48 |
Claims
What is claimed are:
1. A bactericidal/permeability-increasing protein (BPI) deletion
analog consisting of amino acid residues 10 through 193 of mature
human BPI, wherein a cysteine residue at position 132 is replaced
by a different amino acid.
2. The BPI deletion analog of claim 1 wherein the amino acid
replacing said cysteine residue is a non-polar amino acid selected
from the group consisting of alanine and serine.
3. The BPI deletion analog of claim 1 wherein the cysteine residue
at position 132 is replaced by alanine.
4. A polynucleotide encoding the BPI deletion analog of claim
1.
5. A polynucleotide encoding the BPI deletion analog of claim
3.
6. The polynucleotide of claim 4 further comprising the
twenty-seven amino acid leader sequence of BPI.
7. The polynucleotide of claim 4 which is a DNA.
8. An expression vector comprising the DNA according to claim
7.
9. A host cell stably transformed or transfected with the DNA of
claim 7 in a manner allowing expression in said host cell of said
polypeptide deletion analog.
10. A eukaryotic host cell according to claim 9.
11. The host cell of claim 10 which is a CHO cell.
12. A method for producing a BPI deletion analog polypeptide
comprising growing a host cell according to claim 9 in a suitable
culture medium and isolating said polypeptide from said host cell
or said culture medium.
13. The polypeptide product of the method of claim 12.
14. A composition comprising the BPI deletion analog of claim 1 and
a pharmaceutically-acceptable diluent, adjuvant, or carrier.
15. A composition comprising the BPI deletion analog of claim 3 and
a pharmaceutically-acceptable diluent, adjuvant, or carrier.
16. A composition comprising the BPI deletion analog of claim 13
and a pharmaceutically-acceptable diluent, adjuvant, or
carrier.
17. An improved method of administering a BPI protein product to a
subject comprising administering the composition of claim 14 to
said subject.
18. An improved method of administering a BPI protein product to a
subject comprising administering the composition of claim 15 to
said subject.
19. An improved method of administering a BPI protein product to a
subject comprising administering the composition of claim 16 to
said subject.
Description
[0001] This application is a continuation-in-part of U.S. Ser. No.
09/099,725 filed Jun. 19, 1998.
BACKGROUND OF THE INVENTION
[0002] The present invention provides preparations of novel
biologically active deletion analogs of
bactericidal/permeability-increasing protein (BPI) characterized by
improved stability and homogeneity as well as by enhanced in vivo
activity, and pharmaceutical compositions containing the same.
[0003] BPI is a protein isolated from the granules of mammalian
polymorphonuclear leukocytes (PMNs or neutrophils), which are blood
cells essential in the defense against invading microorganisms. BPI
is known to bind to lipopolysaccharide, a major component of the
outer membrane of gram-negative bacteria that stimulates a potent
inflammatory response which can lead to septic shock. Human BPI
protein has been isolated from PMNs by acid extraction combined
with either ion exchange chromatography [Elsbach, J. Biol. Chem.,
254: 11000 (1979)] or E. coli affinity chromatography [Weiss, et
al., Blood, 69: 652 (1987)]. BPI obtained in such a manner is
referred to herein as natural BPI and has been shown to have potent
bactericidal activity against a broad spectrum of gram-negative
bacteria. The molecular weight of human BPI is approximately 55,000
daltons (55 kD). The amino acid sequence of the entire human BPI
protein and the nucleic acid sequence of DNA encoding the protein
have been reported in FIG. 1 of Gray et al., J. Biol. Chem., 264:
9505 (1989), incorporated herein by reference. The Gray et al.
amino acid sequence is set out in SEQ ID NO: 1 hereto. U.S. Pat.
No. 5,198,541, the disclosure of which is incorporated herein by
reference, discloses recombinant genes encoding, and methods for
expression of, BPI proteins including recombinant BPI holoprotein,
referred to as rBPI, and recombinant fragments of BPI.
[0004] A proteolytic N-terminal fragment of BPI having a molecular
weight of about 25 kD has an amphipathic character, containing
alternating hydrophobic and hydrophilic regions. This N-terminal
fragment of human BPI possesses the anti-bacterial activity of the
naturally-derived 55 kD human BPI holoprotein. [Ooi et al., J. Bio.
Chem., 262: 14891-14894 (1987)]. In contrast to the N-terminal
portion, the C-terminal region of the isolated human BPI protein
displays only slightly detectable anti-bacterial activity against
gram-negative organisms. [Ooi et al., J. Exp. Med., 174: 649
(1991).] An N-terminal BPI fragment of approximately 23 kD,
referred to as "rBPI.sub.23," has been produced by recombinant
means and also retains anti-bacterial activity against
gram-negative organisms. [Gazzano-Santoro et al., Infect. Immun.
60: 4754-4761 (1992).] An N-terminal analog of BPI, rBPI.sub.21,
has been produced as described in Horwitz et al., Protein
Expression Purification, 8: 28-40 (1996).
[0005] The bactericidal effect of BPI has been reported to be
highly specific to gram-negative species, e.g., in Elsbach and
Weiss, Inflammation: Basic Principles and Clinical Correlates, eds.
Gallin et al., Chapter 30, Raven Press, Ltd. (1992). This reported
target cell specificity was believed to be the result of the strong
attraction of BPI for lipopolysaccharide (LPS), which is unique to
the outer membrane (or envelope) of gram-negative organisms.
Although BPI was commonly thought to be non-toxic for other
microorganisms, including yeast, and for higher eukaryotic cells,
it has recently been discovered, as discussed infra, that BPI
protein products, exhibit activity against gram-positive bacteria,
mycoplasma, mycobacteria, fungi, protozoa, and chlamydia.
[0006] The precise mechanism by which BPI kills gram-negative
bacteria is not yet completely elucidated, but it is believed that
BPI must first bind to the surface of the bacteria through
electrostatic and hydrophobic interactions between the cationic BPI
protein and negatively charged sites on LPS. LPS has been referred
to as "endotoxin" because of the potent inflammatory response that
it stimulates, i.e., the release of mediators by host inflammatory
cells which may ultimately result in irreversible endotoxic shock.
BPI binds to lipid A, reported to be the most toxic and most
biologically active component of LPS.
[0007] In susceptible gram-negative bacteria, BPI binding is
thought to disrupt LPS structure, leading to activation of
bacterial enzymes that degrade phospholipids and peptidoglycans,
altering the permeability of the cell's outer membrane, and
initiating events that ultimately lead to cell death. [Elsbach and
Weiss (1992), supra]. BPI is thought to act in two stages. The
first is a sublethal stage that is characterized by immediate
growth arrest, permeabilization of the outer membrane and selective
activation of bacterial enzymes that hydrolyze phospholipids and
peptidoglycans. Bacteria at this stage can be rescued by growth in
serum albumin supplemented media [Mannion et al., J. Clin. Invest.,
85: 853-860 (1990)]. The second stage, defined by growth inhibition
that cannot be reversed by serum albumin, occurs after prolonged
exposure of the bacteria to BPI and is characterized by extensive
physiologic and structural changes, including apparent damage to
the inner cytoplasmic membrane.
[0008] Initial binding of BPI to LPS leads to organizational
changes that probably result from binding to the anionic groups of
LPS, which normally stabilize the outer membrane through binding of
Mg.sup.++ and Ca.sup.++. Attachment of BPI to the outer membrane of
gram-negative bacteria produces rapid permeabilization of the outer
membrane to hydrophobic agents such as actinomycin D. Binding of
BPI and subsequent gram-negative bacterial killing depends, at
least in part, upon the LPS polysaccharide chain length, with long
O-chain bearing, "smooth" organisms being more resistant to BPI
bactericidal effects than short O-chain bearing, "rough" organisms
[Weiss et al., J. Clin. Invest. 65: 619-628 (1980)]. This first
stage of BPI action, permeabilization of the gram-negative outer
envelope, is reversible upon dissociation of the BPI, a process
requiring high concentrations of divalent cations and synthesis of
new LPS [Weiss et al., J. Immunol. 132: 3109-3115 (1984)]. Loss of
gram-negative bacterial viability, however, is not reversed by
processes which restore the envelope integrity, suggesting that the
bactericidal action is mediated by additional lesions induced in
the target organism and which may be situated at the cytoplasmic
membrane (Mannion et al., J. Clin. Invest. 86: 631-641 (1990)).
Specific investigation of this possibility has shown that on a
molar basis BPI is at least as inhibitory of cytoplasmic membrane
vesicle function as polymyxin B (In't Veld et al., Infection and
Immunity 56: 1203-1208 (1988)) but the exact mechanism as well as
the relevance of such vesicles to studies of intact organisms has
not yet been elucidated.
[0009] BPI protein products (which include naturally and
recombinantly produced BPI protein; natural, synthetic, and
recombinant biologically active polypeptide fragments of BPI
protein; biologically active polypeptide variants of BPI protein or
fragments thereof, including hybrid fusion proteins and dimers;
biologically active polypeptide analogs of BPI protein or fragments
or variants thereof, including cysteine-substituted analogs; and
BPI-derived peptides) have been demonstrated to have a variety of
beneficial activities. BPI protein products are known to be
bactericidal for gram-negative bacteria, as described in U.S. Pat.
Nos. 5,198,541 and 5,523,288, both of which are incorporated herein
by reference. BPI protein products are also known to enhance the
effectiveness of antibiotic therapy in gram-negative bacterial
infections, as described in U.S. Pat. No. 5,523,288 and
corresponding International Publication No. WO 95/08344
(PCT/US94/11225), which are incorporated herein by reference. BPI
protein products are also known to be bactericidal for
gram-positive bacteria and mycoplasma, and to enhance the
effectiveness of antibiotics in gram-positive bacterial infections,
as described in U.S. Pat. No. 5,578,572 and corresponding
International Publication No. WO 95/19180 (PCT/US95/00656), which
are incorporated herein by reference. BPI protein products are
further known to exhibit anti-fungal activity, and to enhance the
activity of other anti-fungal agents, as described in U.S. Pat. No.
5,627,153 and corresponding International Publication No. WO
95/19179 (PCT/US95/00498), and further as described for anti-fungal
peptides in co-owned, co-pending U.S. application Ser. No.
08/621,259 filed Mar. 21, 1996, which is in turn a
continuation-in-part of U.S. application Ser. No. 08/504,841 filed
Jul. 20, 1994 and corresponding International Publication Nos. WO
96/08509 (PCT/US95/09262) and WO 97/04008 (PCT/US96/03845), all of
which are incorporated herein by reference. BPI protein products
are further known to exhibit anti-protozoan activity, as described
in U.S. Pat. No. 5,646,114 and corresponding International
Publication No. WO 96/01647 (PCT/US95/08624), all of which are
incorporated herein by reference. BPI protein products are known to
exhibit anti-chlamydial activity, as described in co-owned,
co-pending U.S. application Ser. No. 08/694,843 filed Aug. 9, 1996
and corresponding International Publication No. WO 98/06415
(PCT/US97/13810), all of which are incorporated herein by
reference. Finally, BPI protein products are known to exhibit
anti-mycobacterial activity, as described in co-owned, co-pending
U.S. application Ser. No. 08/626,646 filed Apr. 1, 1996, which is
in turn a continuation of U.S. application Ser. No. 08/285,803
filed Aug. 14, 1994, which is in turn a continuation-in-part of
U.S. application Ser. No. 08/031,145 filed Mar. 12, 1993 and
corresponding International Publication No. WO94/20129
(PCT/US94/02463), all of which are incorporated herein by
reference.
[0010] The effects of BPI protein products in humans with endotoxin
in circulation, including effects on TNF, IL-6 and endotoxin are
described in U.S. Pat. Nos. 5,643,875 and 5,753,620 and
corresponding International Publication No. WO 95/19784
(PCT/US95/01151), all of which are incorporated herein by
reference.
[0011] BPI protein products are also known to be useful for
treatment of specific disease conditions, such as meningococcemia
in humans (as described in co-owned, co-pending U.S. application
Ser. No. 08/644,287 filed May 10, 1996 and corresponding
International Publication No. WO 97/42966 (PCT/US97/08016), which
are incorporated herein by reference), hemorrhagic trauma in
humans, (as described in co-owned, co-pending U.S. application Ser.
No. 08/862,785, a continuation-in-part of U.S. Ser. No. 08/652,292
filed May 23, 1996, now U.S. Pat. No. 5,756,464, and corresponding
International Publication No. WO 97/44056 (PCT/US97/08941), all of
which are incorporated herein by reference), burn injury (as
described in U.S. Pat. No. 5,494,896 and corresponding
International Publication No. WO 96/30037 (PCT/US96/02349), both of
which are incorporated herein by reference), ischemia/reperfusion
injury (as described in U.S. Pat. No. 5,578,568, incorporated
herein by reference), and liver resection (as described in
co-owned, co-pending U.S. application Ser. No. 08/582,230 filed
Mar. 16, 1998 which is a continued prosecution application of the
same serial no. filed Jan. 3, 1996, which is in turn a continuation
of U.S. application Ser. No. 08/318,357 filed Oct. 5, 1994, which
is in turn a continuation-in-part of U.S. Application Serial No.
08/132,510 filed Oct. 5, 1993, and corresponding International
Publication No. WO 95/10297 (PCT/US94/11404), all of which are
incorporated herein by reference).
[0012] BPI protein products are also known to neutralize the
anti-coagulant activity of exogenous heparin, as described in U.S.
Pat. No. 5,348,942, incorporated herein by reference, as well as to
be useful for treating chronic inflammatory diseases such as
rheumatoid and reactive arthritis, as described in U.S. Pat. No.
5,639,727, incorporated herein by reference, and for inhibiting
angiogenesis and for treating angiogenesis-associated disorders
including malignant tumors, ocular retinopathy and endometriosis,
as described in co-owned, co-pending U.S. application Ser. Nos.
08/435,855, 08/466,624 and 08/466,826, and corresponding
International Publication No. WO 94/20128 (PCT/US94/02401), all of
which are incorporated herein by reference.
[0013] BPI protein products are also known for use in
antithrombotic methods, as described in U.S. Pat. No. 5,741,779 and
corresponding International Publication No. WO97/42967
(PCT/US97/08017), which are incorporated herein by reference.
[0014] U.S. Pat. Nos. 5,420,019 and 5,674,834 and corresponding
International Publication No. WO94/18323 (PCT/US94/01235), all of
which are incorporated herein by reference, discloses that the
replacement of the cysteine residue at amino acid position 132 or
135 with another amino acid renders the resulting BPI polypeptide
resistant to dimerization and cysteine adduct formation. It also
discloses that terminating the N-terminal BPI fragment at BPI amino
acid position 193 resulted in an expression product with reduced
carboxy-terminal heterogeneity.
[0015] Of interest is the report in Capodici and Weiss, J.
Immunol., 156: 4789-4796 (1996) that the in vitro
transcription/translation products of DNA encoding amino acid
residues 1 through 193 (BPI.sub.1-193) and residues 13 through 193
(BPI.sub.13-193) of mature BPI showed similar LPS-dependent binding
to immobilized LPS.
[0016] There continues to be a need in the art for improved
biologically active BPI protein product preparations, particularly
those with enhanced stability, homogeneity and/or in vivo
biological activity.
SUMMARY OF THE INVENTION
[0017] The present invention provides novel biologically active BPI
deletion analogs and preparations thereof characterized by enhanced
stability and homogeneity, including for example, resistance to
dimerization and cysteine adduct formation and reduced
amino-terminal and carboxy-terminal heterogeneity of the
recombinant product, as well as by enhanced in vivo biological
activity, properties which render it highly suitable for
therapeutic and diagnostic uses. Novel BPI deletion analogs are the
expression product of DNA encoding amino acid residues 10 through
193 of mature human BPI (SEQ ID NO: 2), in which the cysteine at
position 132 has been replaced with a different amino acid,
preferably a non-polar amino acid such as serine or alanine. In a
preferred embodiment, designated "rBPI(10-193)C132A" or
"rBPI(10-193)ala.sup.132," the cysteine at position 132 is replaced
with an alanine.
[0018] The invention further provides novel purified and isolated
polynucleotide sequences (e.g., DNA or RNA) encoding these BPI
protein products; materials and methods for their recombinant
production, including vectors and host cells comprising the DNA;
improved stable pharmaceutical compositions comprising these BPI
protein products; and improved treatment methods using these
compositions, either alone or concurrently administered with other
therapeutic agents. Also contemplated is the use of the BPI
deletion analogs of the invention in manufacture of a medicament
for treating a subject that would benefit from administration of
BPI protein product.
[0019] Numerous additional aspects and advantages of the invention
will become apparent to those skilled in the art upon considering
the following detailed description of the invention, which
describes the presently preferred embodiments thereof.
BRIEF DESCRIPTION OF THE DRAWINGS
[0020] FIG. 1 depicts the elevation in blood pressure, measured as
area under the curve (AUC) occurring after administration of either
rBPI(10-193)C132A or rBPI.sub.21.
DETAILED DESCRIPTION OF THE INVENTION
[0021] The present invention provides novel BPI deletion analogs
consisting of amino acid residues 10 through 193 of mature human
BPI (set forth in SEQ ID NO: 2) wherein the cysteine residue at BPI
amino acid position 132 is replaced by another amino acid,
preferably a non-polar amino acid such as serine or alanine. A
preferred embodiment, in which the cysteine at position 132 is
replaced with an alanine, has been designated rBPI(10-193)C132A or
rBPI(10-193)ala.sup.132.
[0022] The BPI protein product rBPI.sub.21 is the expression
product of DNA encoding amino acid residues 1 to 193 of mature
human BPI wherein the cysteine at residue number 132 is substituted
with alanine, described in U.S. Pat. No. 5,420,019. Changes in the
fermentation processes used to produce rBPI.sub.21 by recombinant
methods that achieved higher cell densities and higher rBPI.sub.21
titers also resulted in an apparent increase in amino-terminal
heterogeneity of the purified product. In some fermentation runs,
up to about 20% of the purified product was observed to be a
species with amino acids 10-193 of BPI, rather than the encoded
1-193 amino acids. SDS-PAGE gels of 500-liter fermentor samples
over the course of a fermentation run showed that this 10-193
species appeared in the last 2-3 days of the run, with the greatest
amount appearing on the day of harvest. Further investigation
revealed that incubation of rBPI.sub.21 with a CHO-K1 cell
homogenate yielded a digested product, suggesting that protease
activity associated with the cells was involved. To simulate
protease activity in a controlled manner, rBPI.sub.21 was incubated
with aminopeptidase M and elastase. The rBPI.sub.21 was resistant
to aminopeptidase M digestion, but elastase rapidly converted the
rBPI.sub.21 into 40% BPI(8-193) and 60% BPI(10-193).
[0023] As described herein, stable homogeneous preparations of
rBPI(10-193)C132A were produced proteolytically and by recombinant
methods. The protein was purified and was tested for biological
activity. Experiments were performed to compare rBPI(10-193)C132A
to rBPI.sub.21 in several in vitro biological assays, two different
animal efficacy models and in pharmacokinetic and toxicology
studies. As described in Examples 5-7, rBPI(10-193)C132A and
rBPI.sub.21 had similar in vitro activities when compared in radial
diffusion and broth microdilution bactericidal assays with
Escherichia coli J5, a radial diffusion assay with an L-form of
Staphylococcus aureus, a competition binding assay with E. coli J5
LPS, and in LPS neutralization assays with RAW and THP1 cells.
Additional experiments described in Example 5 showed that
rBPI(10-193)C132A appeared to be approximately twice as potent as
rBPI.sub.21 in an LPS binding assay using rate nephelometry. As
described in Example 8, purified rBPI(10-193)C132A and rBPI.sub.21
had similar toxicity profiles in a GLP toxicology study in rats at
doses up to 120 mg/kg/day for three days and similar
pharmacokinetics in rats at a dose of 2 mg/kg. Experiments
described in Example 8 also showed that in a mouse endotoxin
challenge model, rBPI(10-193)C132A appeared to be at least two-fold
more potent than rBPI.sub.21 in two studies whereas in a mouse
model of lethal bacteremia, rBPI(10-193)C132A and rBPI.sub.21 were
similarly potent. In additional in vivo experiments in conscious
rats, doses of 40 and 50 mg/kg of infused rBPI.sub.21 caused
significant transient decreases in blood pressure relative to the
vehicle control, while the same doses of rBPI(10-193)C132A did not
result in a statistically significant transient decrease in blood
pressure relative to control. Thus, infusion of rBPI(10-193)C132A
appears to provide a reduction in an adverse effect in blood
pressure compared with infusion of rBPI.sub.21.
[0024] The invention further contemplates fusion of
rBPI(10-193)C132A with at least a portion of at least one other
polypeptide. Examples of such hybrid fusion proteins are described
in U.S. Pat. No. 5,643,570 and corresponding International
Publication No. WO 93/23434 (PCT/US93/04754), which are all
incorporated herein by reference and include hybrid fusion proteins
comprising, at the amino-terminal end, a BPI protein or a
biologically active fragment thereof and, at the carboxy-terminal
end, at least one constant domain of an immunoglobulin heavy chain
or allelic variant thereof.
[0025] The invention additionally contemplates purified and
isolated polynucleotide sequences (e.g., DNA or RNA) encoding the
novel BPI deletion analogs or fusion proteins of the present
invention; expression vectors containing such polynucleotides,
preferably operatively linked to an endogenous or heterologous
expression control sequence; prokaryotic or eukaryotic host cells
stably transfected or transformed with a DNA or vector of the
present invention; and methods for the recombinant production of
the novel deletion analog BPI protein products of the present
invention, e.g., methods in which a host cell is grown in a
suitable nutrient medium and the deletion analog BPI protein
product is isolated from the cell or the medium. Such
polynucleotide sequences or vectors may optionally encode the
27-amino acid BPI leader sequence and the mouse light chain
polyadenylation signal.
[0026] The recombinantly produced novel BPI deletion analog of the
present invention may be produced according to the methods
described in U.S. Pat. No. 5,439,807 and corresponding
International Publication No. WO 93/23540 (PCT/US93/04752), which
are all incorporated herein by reference. U.S. Pat. No. 5,439,807
discloses methods for the purification of recombinant BPI protein
products expressed in and secreted from genetically transfected
mammalian host cells in culture, and discloses how one may produce
large quantities of recombinant BPI products suitable for
incorporation into stable, homogeneous pharmaceutical
preparations.
[0027] The present invention further provides improved stable
pharmaceutical compositions comprising the novel BPI deletion
analogs and improved treatment methods using these compositions,
either alone or concurrently administered with other therapeutic
agents. It is contemplated that such compositions may be utilized
in any of the therapeutic uses known for BPI protein products,
including those discussed supra.
[0028] The administration of BPI protein products in general,
including BPI deletion analogs, is preferably accomplished with a
pharmaceutical composition comprising a BPI protein product and a
pharmaceutically acceptable diluent, adjuvant, or carrier. The BPI
protein product may be administered without or in conjunction with
known surfactants, other chemotherapeutic agents or additional
known anti-chlamydial agents. A stable pharmaceutical composition
containing BPI protein products (e.g., rBPI.sub.23) comprises the
BPI protein product at a concentration of 1 mg/ml in citrate
buffered saline (5 or 20 mM citrate, 150 mM NaCl, pH 5.0)
comprising 0.1% by weight of poloxamer 188 (Pluronic F-68, BASF,
Parsippany, N.J.) and 0.002% by weight of polysorbate 80 (Tween 80,
ICI Americas Inc., Wilmington, Del. or JT Baker, Phillipsburg,
N.J.). Another stable pharmaceutical composition containing BPI
protein products (e.g., rBPI.sub.21) comprises the BPI protein
product at a concentration of 2 mg/ml in 5 mM citrate, 150 mM NaCl,
0.2% poloxamer 188 and 0.002% polysorbate 80. Such preferred
combinations are described in U.S. Pat. Nos. 5,488,034 and
5,696,090 and corresponding International Publication No. WO
94/17819 (PCT/US94/01239), the disclosures of all of which are
incorporated herein by reference. As described in U.S. application
Ser. No. 08/586,133 filed Jan. 12, 1996, which is in turn a
continuation-in-part of U.S. application Ser. No. 08/530,599 filed
Sep. 19, 1995, which is in turn a continuation-in-part of U.S.
application Ser. No. 08/372,104 filed Jan. 13, 1995, and
corresponding International Publication No. WO96/21436
(PCT/US96/01095), all of which are incorporated herein by
reference, other poloxamer formulations of BPI protein products
with enhanced activity may be utilized.
[0029] Therapeutic compositions comprising BPI protein product may
be administered systemically or topically. Systemic routes of
administration include oral and parenteral routes, including
intravenous, intramuscular or subcutaneous injection (including
into a depot for long-term release), intraocular and retrobulbar,
intrathecal, intraperitoneal (e.g. by intraperitoneal lavage),
intrapulmonary (using powdered drug, or an aerosolized or nebulized
drug solution), or transdermal. Improved aerosolized formulations
are described in co-owned, co-pending U.S. application Ser. No.
08/962,217 filed Oct. 31, 1997 and corresponding International
Publication No. WO 98/19694 (PCT/US97/19850), which are both
incorporated herein by reference.
[0030] When given parenterally, BPI protein product compositions
are generally injected in doses ranging from 1 .mu.g/kg to 100
mg/kg per day, preferably at doses ranging from 0.1 mg/kg to 20
mg/kg per day, more preferably at doses ranging from 1 to 20
mg/kg/day and most preferably at doses ranging from 2 to 10
mg/kg/day. The treatment may continue by continuous infusion or
intermittent injection or infusion, at the same, reduced or
increased dose per day for, e.g., 1 to 3 days, and additionally as
determined by the treating physician. When administered
intravenously, BPI protein products are preferably administered by
an initial brief infusion followed by a continuous infusion. The
preferred intravenous regimen is a 1 to 20 mg/kg brief intravenous
infusion of BPI protein product followed by a continuous
intravenous infusion at a dose of 1 to 20 mg/kg/day, continuing for
up to one week. A particularly preferred intravenous dosing regimen
is a 1 to 4 mg/kg initial brief intravenous infusion followed by a
continuous intravenous infusion at a dose of 1 to 4 mg/kg/day,
continuing for up to 72 hours.
[0031] Topical routes include administration in the form of salves,
creams, jellies, ophthalmic drops or ointments (as described in
co-owned, co-pending U.S. application Ser. No. 08/557,289 filed
Nov. 14, 1995 and U.S. Pat. No. 5,686,414 and corresponding
International Publication Nos. WO 97/17990 (PCT/US96/18632) and WO
97/17989 (PCT/US96/18416), all of which are incorporated herein by
reference), ear drops, suppositories, irrigation fluids (for, e.g.,
irrigation of wounds) or medicated shampoos. For example, for
topical administration in drop form, about 10 to 200 .mu.L of a BPI
protein product composition may be applied one or more times per
day as determined by the treating physician.
[0032] Those skilled in the art can readily optimize effective
dosages and administration regimens for therapeutic compositions
comprising BPI protein product, as determined by good medical
practice and the clinical condition of the individual patient.
[0033] Other aspects and advantages of the present invention will
be understood upon consideration of the following illustrative
examples. Example 1 addresses the construction of an expression
vector, pING1742, encoding rBPI(10-193)C132A. Example 2 addresses
transformation of CHO cells with pING1742 and selection of the
highest producing clones secreting rBPI(10-193)C132A. Example 3
addresses the production and purification of rBPI(10-193)C132A in
2-L and 500-L fermenters. Example 4 addresses the biochemical
characterization of rBPI(10-193)C132A and rBPI.sub.21. Examples 5,
6 and 7 respectively address the in vitro LPS-binding activity in a
competition binding assay and in an assay measuring rate of complex
formation using rate nephelometry, bactericidal activity, and LPS
neutralization activity of rBPI(10-193)C132A as compared to
rBPI.sub.21. Example 8 addresses the in vivo activity of
rBPI(10-193)C132A.
EXAMPLE 1
Construction of Expression Vector pING1742
[0034] The rBPI(10-193)C132A expression vector, pING1742, was
constructed as follows. The expression vector pING4155 was first
constructed by ligating a BamHI-BsaI fragment containing the neo
gene from pING3174 with a BsaI-XhoI fragment containing the CMV
promoter and rBPI.sub.21 gene from pING4144 and an XhoI-BamHI
fragment containing the mouse (kappa) light chain 3' untranslated
region from pING4537 (pING3174, pING4144 and pING4537 are described
in U.S. Pat. No. 5,420,019, incorporated by reference). The
resulting pING4155 vector contains the gene encoding rBPI.sub.21
fused to the human IgG enhancer, the human CMV promoter and the
mouse (kappa) light chain 3' untranslated region. It also contains
the neo gene encoding neomycin phosphotransferase, for selection of
transfectants resistant to the antibiotic Geneticin.RTM.
(G418).
[0035] The vector pING1732 was produced by deleting the 0.7 kbp
HindIII-HindIII fragment of pING4155 containing the human Ig
enhancer. Then, the 27 nucleotides encoding amino acids 1 through 9
of the mature portion of rBPI.sub.21 were deleted from pINGI 732 by
overlap PCRm utagenesis using the following primers:
1 (SEQ ID NO: 3) Primer 1: 5'-CTGCTCTAAAAGCTGCTGCAG-3' (SEQ ID NO:
4) Primer 2: 5'-CCAGGCCCTTCTGGGAGGCCGCTGT- CACGGCGG-3' (SEQ ID NO:
5) Primer 3: 5'-GCCGTGACAGCGGCCTCCCAGAAGGGCCTGGAC-3' (SEQ ID NO: 6)
Primer 4: 5'-CTGGGAACTGGGAAGCTG-3'
[0036] Overlapping complementary primers 2 and 3 incorporated the
27 bp deletion of nucleotides encoding amino acids 1 through 9,
while primers 1 and 4 encoded nucleotides immediately upstream and
downstream, respectively, of unique SalI and EcoRI sites in
pING1732. First, fragments were obtained by PCR amplification using
the combination of oligonucleotide primers 1 and 3, and primers 2
and 4. After these individual fragments were obtained, they were
annealed, extended and re-amplified using primers 1 and 4. This
amplified fragment was then digested with SalI and EcoR[and cloned
into SalI-EcoRI-digested pING1732 to generate the plasmid
pING1742.
[0037] To confirm that no mutations had occurred during PCR, the
SalI-EcoRI region from pING1742 was sequenced. No changes were
observed in the mature coding region for BPI. However, a two
base-pair change (ACC->GCT) was found in DNA encoding the signal
sequence, which resulted in the conversion of a Thr to an Ala at
amino acid position-6 relative to the start of the mature protein
sequence.
EXAMPLE 2
Transformation of CHO Cells with pING1742
[0038] CHO-K1 cells (American Type Culture Collection (ATCC)
Accession No. CCL61) were adapted to growth in serum-free Ex-Cell
301 medium as follows. CHO-K1 cells grown in Ham's F12 medium were
trypsinized, centrifuged and resuspended in Ex-Cell 301 medium.
Cells were grown in a 125-ml flask at 100 rpm and passaged every
two to three days in either a 125-ml or 250-ml flask.
[0039] These Ex-Cell 301-adapted CHO cells were transfected by
electroporation with pING1742. Prior to transfection, pING1742 was
digested with NotI, which linearizes the plasmid. Following a
48-hour recovery, cells were plated at approximately 10.sup.4
cells/well into 96-well plates containing Ex-Cell 301 medium
supplemented with 0.6 mg/mL G418 (Life Technologies, Gaithersburg,
Md.). At approximately 2 weeks, supernatants from approximately 250
wells containing single colonies were screened by ELISA for the
presence of BPI-reactive protein using an anti-BPI monoclonal
antibody.
[0040] Fifteen clones having the highest expression levels were
transferred to 24-well plates containing Ex-Cell 301 medium. To
screen for productivity, the cells were grown in 24-well plates
containing Ex-Cell medium supplemented with 2% FBS and 40 .mu.L
sterile S-Sepharose beads for 10 days, after which the beads were
removed, washed with low salt buffer (0.1 M NaCl in 10 mM Na
acetate, pH 4.0) and the BPI eluted with 1.5 M NaCl in the same
buffer. The levels of secreted rBPI(10-193)C132A were determined by
ELISA. Western blot analysis of eluates run on a 12% non-reducing
SDS gel revealed a prominent band which migrated slightly faster
than rBPI.sub.21.
[0041] The top eight producers were transferred to sterile 125 mL
Erlenmeyer flasks and grown in Ex-Cell medium. These cells were
evaluated again for productivity by growing them in flasks
containing Ex-Cell 301 medium supplemented with 2% FBS and 1% (VN)
sterile S-sepharose beads. The rBPI(10-193)C132A was eluted from
the S-Sepharose beads that had been incorporated in the culture
medium and the levels of rBPI(10-193)C132A determined by HPLC.
Clone 139, which was among the highest producers, was chosen for
further growth and product production.
EXAMPLE 3
Production and Purification of rBPI(10-193)C132A
[0042] Large quantities of rBPI(10-193)C132A were produced for
characterization by growing Clone 139 cells in 2-liter research
fermenters (Biolafitte, St. Germain en Laye, France) and then in a
500 liter ABEC fermenter (ABEC, Allentown, Pa.). Protein product
obtained from the 2-liter fermenters was used for the in vitro
studies described below, while product obtained from the 500 liter
fermenter was used for animal toxicology and efficacy studies.
[0043] A. Growth in Two-Liter Fermenters
[0044] Clone 139 cells were passaged in spinner flasks of
increasing volumes containing Ex-Cell medium supplemented with 1%
FBS until sufficient volume and cell density was achieved to
inoculate the 2 liter bioreactors at approximately 2.times.10.sup.5
cells/mL. Cells were grown in three 2-liter fermenters in Ex-Cell
medium supplemented with 1% FBS, at 37.degree. C., pH 7.2, 150 rpm
with dissolved oxygen maintained at 5-10%. Large sterile
SP-Sepharose beads (Pharmacia and Upjohn, Piscataway, N.J.) were
added at 1.5% (VN). The initial glucose level was approximately 3.5
g/L and glucose was pulsed daily to 3 g/L during the course of the
run. The fermentation was terminated at 238 hours, at which time
the cell viabilities were from 63%, 80% and 84%.
[0045] Following fermentation, the beads from each fermenter were
harvested, allowed to settle, and washed several times with 10 mM
Na phosphate/0.15M NaCl, pH 7.0, to remove cellular components and
weakly bound impurities from the beads. The washed beads were
packed into a column, washed with 10 mM Na phosphate, 0.25 M NaCl,
pH 7.0, and eluted with the same buffer containing 0.8 M NaCl, 5 mM
glycine. The eluate was then diluted with three volumes of sterile
water for injection (WFI), loaded onto a CM-spherodex column
(Sepracor, Marlborough, Mass.) and washed with 10 mM Na phosphate,
0.25 M NaCl, pH 7.0, followed by 20 mM Na acetate, 0.2 M NaCl, pH
4.0, followed by 20 mM Na acetate, 0.3 M NaCl, pH 4.0, and sample
was eluted at 1.0 M NaCl in the same buffer. Following
concentration on a Centricon membrane with a 10,000 MW cutoff
(Amicon, Beverly, Mass.), the eluate from the CM column was loaded
onto a Sephacryl S-100 column (Pharmacia and Upjohn) equilibrated
with 5 mM Na citrate, 0.15 M NaCl, pH 5.0. Fractions containing
rBPI(10-193)C132A identified by absorbance at 280 nm were pooled,
concentrated on an Amicon filter to 1.9 mg/mL and formulated with
0.002% polysorbate 80 (JT Baker, Phillipsburg, N.J.), 0.2%
poloxamer 188 (Pluronic F-68, BASF, Parsippany, N.J.). The final
preparation was filter sterilized using a 0.2 .mu.m filter.
[0046] B. Growth in 500-Liter Fermenter
[0047] Clone 139 cells were passaged in fetuin-free Ex-Cell medium
with 1% FBS in a series of spinner flasks of increasing volumes to
provide inoculum for the 35L Bellco spinner flask (Bellco Glass,
Vineland, N.J.), which in turn provided the inoculum for the 500
liter ABEC fermenter. Cells were grown in complete Ex-Cell medium
without fetuin but supplemented with 1% FBS, additional glucose (to
10 g/L) and glutamine (to 10 mM). The fermenter was operated in a
fed-batch mode with one 0.5% Primatone RL supplement pulse and one
glucose/glutamine pulse added during the run. Five to six liters of
large SP-Sepharose beads were added 24 hours after the 500 liter
fermenter was inoculated. The pH was controlled manually with 10%
sodium bicarbonate to pH 7.0, oxygen was controlled at 5% and
temperature at 37.degree. C. Agitation was maintained at 25 rpm
with two three-blade paddle impellers. The fermentation run was
terminated at 184 hours, at which time the cell viability was
90%.
[0048] As described above for the 2-liter fermentation, the beads
were allowed to settle following fermentation and then washed
several times with low salt (0.1M) phosphate buffer. The steps for
this purification were similar to those described above for the
2-liter samples except that a pH 3.0 viral inactivation step was
included after elution from the S-Sepharose beads and a second
CM-spherodex column was included as a concentration step. F or the
second CM column, the eluate was diluted with three volumes of WFI,
the pH adjusted to 5.0, the column was equilibrated and washed with
20 mM Na acetate, 0.3 M NaCl, pH 5.0 and the sample was eluted at
1.0 M NaCl in the same buffer. The rBPI(10-193)C132A was eluted
from the Sephacryl S-100 column in 5 mM Na citrate, 0.15 M NaCl, pH
5.0, adjusted to 2 mg/mL, and filtered through a 0.2 .mu.m filter.
The rBPI(10-193)C132A w as then formulated with 0.002% polysorbate
80, 0.2% poloxamer 188, sterile filtered, and filled into 10 mL
Type I glass serum vials.
EXAMPLE 4
Biochemical Characterization of rBPI(10-193)C132A
[0049] A. Protein from the 2-Liter Fermentations
[0050] The purified rBPI(10-193)C132A product from Example 3 was
observed to be a single band that migrated slightly faster on SDS
polyacrylamide gel electrophoresis (SDS-PAGE) than the rBPI.sub.21
band, consistent with the deletion of nine N-terminal amino acids
from rBPI.sub.21. Sequence analysis demonstrated that the
rBPI(10-193)C132A contained the predicted N-terminal sequence of
SQKGLDYASQQGTAALQKEL. On mass spectroscopy analysis (ESI-MS) two
components were observed, one with a mass of 20,470 daltons, which
was consistent with the predicted mass of 20,472 daltons for
rBPI(10-193)C132A, and a second with a mass of 20,255 daltons,
consistent with the predicted mass of 20,258 daltons for
rBPI(10-191). The ion-exchange HPLC profiles (Hewlett-Packard,
Model 1050, Palo Alto, Calif.) of rBPI(10-193)C132A and rBPI.sub.2,
both exhibited single peaks with similar retention times.
[0051] B. Protein from the 500-Liter Fermentation
[0052] On SDS-PAGE, the rBPI(10-193)C132A was a single band that
migrated slightly faster than the rBPI.sub.21 band. On mass
spectroscopy, there was a major component with a mass of 20,471
daltons, which is consistent with the predicted mass of 20,474 Da
for rBPI(10-193)C132A), and two minor components with a mass of
20,668 daltons, which is consistent with addition of
N-Acetylhexosamine (predicted mass 20,677 daltons) and a mass of
20,843 daltons, which is consistent with addition of
N-Acetylhexosamine plus hexose (predicted mass 20,839 daltons). A
similar component with added N-Acetylhexosamine is routinely
observed during production of rBPI.sub.21.
[0053] On reverse phase HPLC (Shimadzu, Kyoto, Japan) both the
rBPI(10-193)C132A and rBPI.sub.21 eluted as one major peak and one
minor peak. However, the rBPI(10-193)C132A peaks eluted slightly
earlier than the corresponding rBPI.sub.21 peaks in the control.
The minor peak in the rBPI(10-193)C132A profile most likely
represents the glycosylated forms identified in the mass spectrum.
The ion-exchange HPLC profiles of rBPI(10-193)C132A and rBPI.sub.21
both exhibited single peaks with similar retention times.
[0054] Tryptic mapping analysis was performed according to
conventional methods. Acetone precipitated rBPI.sub.2, or
rBPI(10-193)C132A was first treated with dithiothreitol (DTT)
followed by iodoacetamide and then with trypsin. The
trypsin-treated product was analyzed by HPLC (Beckman Model 126)
with a C18 column (Beckman Ultrasphere). In rBPI.sub.21, there are
two N-terminal tryptic fragments (Ti and Ala-Ti) which result from
imprecise cleavage of the leader sequence. As predicted, the
tryptic map of the rBPI(10-193)C132A was similar to rBPI.sub.21
except that the N-terminal fragments were missing.
EXAMPLE 5
In Vitro LPS-Binding Activity of rBPI(10-193)C132A
[0055] A. In a Competition Binding Assay
[0056] The ability of purified rBPI(10-193)C132A produced according
to Example 3A and rBPI.sub.21 to compete with labeled rBPI.sub.21
for binding to LPS was evaluated in a competition binding assay.
Briefly, a fixed concentration (0.5 nM) of .sup.125I-labeled
rBPI.sub.21 was mixed with unlabeled rBPI.sub.21 or
rBPI(10-193)C132A at dilutions ranging from 5 .mu.M to 0.01 .mu.M
in DMEM containing HEPES buffer and bovine serum albumin (BSA)
[U.S. Biochemicals, Cleveland, Ohio] and 100 .mu.L of the mixture
was added to Immulon-II plate wells pre-coated with 2.5 .mu.g/mL E.
coli J5 LPS (Calbiochem, San Diego, Calif.). The plates were
incubated at 4.degree. C. for 5 hours and washed 3 times with the
DMEM medium. 75 .mu.L of 0.1 N NaOH was added and the bound
1.sup.25]-rBPI.sub.21 was removed and counted. The results
demonstrated that both proteins competed similarly with
radiolabeled rBPI.sub.21.
[0057] B. In an Assay Measuring Rate of Complex Formation
[0058] The LPS binding activity of rBPI(10-193)C132A was compared
to rBPI.sub.21 using rate nephelometry. This approach for
evaluating rBPI.sub.21 binding to LPS measures the rate of increase
of light scattering as a result of LPS-BPI protein product complex
formation in solution. All of the experiments were performed with a
Beckman Array 360 Rate Nephelometer which automatically mixes
samples, measures light scattering and performs rate
calculations.
[0059] Prior experiments using this approach examined optimal LPS
species and concentration, assay specificity, assay reproducibility
and correlation of assay results to bactericidal assays. It was
observed that E. coli J5 LPS and lipid A formed complexes with
rBPI.sub.21 that could be measured in the nephelometer, but E. coli
O111:B4 LPS did not form measurable complexes. Based on results of
these studies, E. coli J5 LPS was chosen for use at a concentration
(in the flow cell) of 49.4 to 61.7 .mu.g/ml, depending on the LPS
lot, in combination with rBPI.sub.21 concentrations (in the flow
cell) from 5 to 30 .mu.g/ml. The optimal rBPI.sub.21 concentration
range, which must be determined for each LPS lot, was from about 15
to 25 .mu.g/ml which represented the most linear portion of the
curve. The optimal range for the aggregation rate (RT) values was
from 700 to 2000. Lower concentrations of rBPI.sub.21 were needed
to achieve the same aggregation rate values when the formulation
buffer was changed to include PLURONIC P103 or when the NaCl
concentration was increased. The addition of either recombinant
lipopolysaccharide binding protein (rLBP.sub.50) which binds to
LPS, or heparin which binds to BPI protein products, inhibited the
formation of rBPI.sub.21-LPS aggregates, demonstrating the
specificity of the interaction. Assay reproducibility was confirmed
by testing multiple lots of BPI and testing the same lot of
rBPI.sub.21 multiple times. Nephelometric analysis of rBPI.sub.21
samples that had been partially inactivated by treatment at
45.degree. C. for one week correlated well with those from broth
microdilution bactericidal assays with E. coli J5 cells.
[0060] Nephelometry experiments comparing rBPI(10-193)C132A and
rBPI.sub.21 were carried out as follows. Sonicated LPS [E. coli J5
LPS Lot No. 30119B from List Biochemicals] and either
rBPI(10-193)C132A or rBPI.sub.21 [both of which were formulated in
0.2% PLURONIC F68 (poloxamer 188), 0.002% TWEEN 80 (polysorbate
80), 5 mM citrate, pH 5.0, 150 mM NaCl] were diluted directly into
a PBS buffer (supplemented with PEG) supplied by Beckman. The LPS
concentration was fixed while the BPI protein product concentration
varied within each experiment. Two concentrations of LPS were
tested: 24.7 and 49.4 .mu.g/ml LPS. Each reaction was initiated by
addition of 600 .mu.l of the PBS-PEG buffer to the flow cell
followed by 42 .mu.l of the BPI protein product dilution. After a
baseline was established, 42 .mu.l of the E. coli J5 LPS solution
was added. After addition of the last component, the nephelometer
measures the rate of complex formation based on the extent of light
scatter. The data were analyzed by dividing the RT values for each
test sample containing a given BPI protein product concentrations
by the corresponding RT values for the standard to generate a
percent of control value. For each BPI protein product
concentration tested, the maximum aggregation rate was determined
and a curve generated. Only points to the left of the maximum value
(point of equivalence) were used for comparative analysis of
various BPI protein product samples. The relative activity of
samples can be measured by comparing the RT values for test and
standard lots in the linear region of the curves. Either a point to
point or curve fit approach can be used.
[0061] In addition to testing purified rBPI(10-193)C132A and
purified rBPI.sub.21 [which contains about 7.8% rBPI(10-193)C132A],
an equal mixture of these proteins as well as a rBPI.sub.21
preparation with 16% rBPI(10-193)C132A was evaluated (at 49.4
.mu.g/ml LPS only). The results demonstrated that at 49.4 .mu.g/ml
LPS, rBPI(10-193)C132A achieved aggregation rates similar to that
of rBPI.sub.21 at an approximately 25% lower concentration. The
rBPI(10-193)C132A also achieved a higher maximum aggregation rate
than that of rBPI.sub.21 at both 24.7 and 49.4 .mu.g/ml LPS. An
equal mix of the two molecules yielded a curve that ran between
rBPI.sub.21 and rBPI(10-193) while the rBPI.sub.21 lots with 7.8%
and 16% 10-193 behaved in an identical manner to each other. A
point to point analysis of the results (LPS at 49.4 .mu.g/ml)
revealed that the rBPI(10-193) was approximately twice as potent as
rBPI.sub.21 in this assay.
EXAMPLE 6
In Vitro Bactericidal Activity of rBPI(10-193)C132A
[0062] All of the assays in this example were conducted with
rBPI(10-193)C132A produced in the 2-liter fermenters according to
Example 3A.
[0063] A. Effect on E. coli in a Radial Diffusion Assay
[0064] This radial diffusion assay compared the bactericidal effect
of purified rBPI(10-193)C132A and rBPI.sub.21 on E. coli J5, which
is a UDP-galactose-4-epimerase "rough" mutant of the smooth strain
E. coli 011B4, and is relatively sensitive to rBPI.sub.21. E. coli
J5 cells (Mannion et al., J. Clin. Invest., 85: 853-860 (1990);
List Biological Laboratories, Campbell, Calif.) were grown to
exponential phase, centrifuged and washed twice in 10 mM Na
phosphate, pH 7.4, and added at a final concentration of
approximately 1.times.10.sup.6 CFU/ml to molten agarose
supplemented with 3% Trypticase Soy Broth (TSB, DIFCO Laboratories,
Detroit, Mich.), 10 mM Na phosphate. Wells of 3 mm diameter were
prepared in the hardened agarose and 5 .mu.L of serially diluted
rBPI.sub.21 or rBPI(10-193)C132A was added to the wells. The plates
were incubated at 37.degree. C. for 3 hours to allow diffusion to
occur, and then a molten agarose overlay containing 6% TSB was
added. The plates were incubated overnight at 37.degree. C. and the
net area of inhibition was plotted vs. concentration. The results
demonstrated that rBPI(10-193)C132A and rBPI.sub.21 behaved in a
similar manner in this assay.
[0065] B. Effect on S. Aureus L-Form in a Radial Diffusion
Assay
[0066] This radial diffusion assay compared the bactericidal effect
of purified rBPI(10-193)C132A and rBPI.sub.21 on the gram-positive
bacteria S. aureus grown as L-forms without their cell walls. As
described in U.S. Pat. No. 5,578,572, incorporated herein by
reference, S. aureus L-form cells were grown to log phase in heart
infusion (HI) broth supplemented with 3.5% NaCl, 10 mM CaCl.sub.2
and 1000 U/mL penicillin G. The cells were diluted to approximately
either 5.times.10.sup.4 or 5.times.10.sup.5 cells/mL in molten 0.8%
agarose containing the NaCl-supplemented HI medium, and 8 ml of the
cell-agarose suspension was poured into 10 cm plates. Wells of 3 mm
diameter were prepared, and 5 uL of serially diluted rBPI.sub.21 or
rBPI(10-193)C132A was added to the wells. The plates were incubated
at 37.degree. C. for 24 hours and the net area of inhibition was
plotted vs. concentration. The results demonstrated that both
rBPI.sub.21 and rBPI(10-193)C132A inhibited growth of the S. aureus
L-forms, at cell densities of about 5.times.10.sup.4 and
5.times.10.sup.5, in a similar fashion in this assay.
[0067] C. Effect on E. coli J5 in a Broth Microdilution Assay
[0068] This broth microdilution assay compared the bactericidal
effect of purified rBPI(10-193)C132A and rBPI.sub.21 on E. coli J5.
E. coli J5 cells were grown overnight in tryptone yeast extract
(TYE) broth and then to logarithmic phase in TEA medium as
previously described in Horwitz et al., Infect. Immun., 63: 522-527
(1995). The cells were inoculated at approximately 1.times.10.sup.4
and 1.times.10.sup.5 cells/mL in heart infusion (HI) broth, and 95
.mu.L was added to 96 well plates. Five .mu.L of various dilutions
of rBPI(10-193)C132A or rBPI.sub.21, prepared in formulation
buffer, was added to each well and the plates were incubated at
37.degree. C. for 24 hours. The results demonstrated that
rBPI(10-193)C132A and rBPI.sub.21 have similar activities in these
assays.
EXAMPLE 7
In Vitro LPS Neutralization Activity of rBPI(10-193)C132A
[0069] The assay in section A of this example was conducted with
rBPI(10-193)C132A produced in the 2-liter fermenters according to
Example 3A, while the assay in section B of this example was
conducted with rBPI(10-193)C132A produced in the 500-liter
fermenter according to Example 3B.
[0070] A. Activity in a RAW Cell Proliferation Assay
[0071] The RAW cell proliferation assay was used to compare the in
vitro LPS neutralization activity of rBPI.sub.21 and
rBPI(10-193)C132A. In this assay, the LPS inhibits the
proliferation of RAW cells, and rBPI.sub.21 neutralizes this effect
of LPS.
[0072] Mouse RAW 264.7 cells (ATCC Accession No. T1B71), maintained
in RPMI 1640 medium (GIBCO), supplemented with 10 mM HEPES buffer
(pH 7.4), 2 mM L-glutamine, penicillin (100 U/mL), streptomycin
(100 .mu.g/mL), 0.075% sodium bicarbonate, 0.15M 2-mercaptoethanol
and 10% fetal bovine serum (Hyclone, Inc., Logan, Utah), were first
induced by incubation in the presence of 50 U/mL recombinant mouse
y-interferon (Genzyme, Cambridge, Mass.) for 24 hours prior to
assay. Induced cells were then mechanically collected and
centrifuged at 500.times.g at 4.degree. C. and then resuspended in
50 mL RPMI 1640 medium (without supplements), re-centrifuged and
again resuspended in RPMI 1640 medium (without supplements). The
cells were counted, their concentration adjusted to
2.times.10.sup.5 cells/mL and 100 .mu.L aliquots were added to each
well of a 96-well plate.
[0073] The cells were then incubated for about 15 hours with E.
coli O113 LPS (Control Standard, Assoc. of Cape Code, Woods Hole,
Mass.), which was added in 100 .mu.L/well aliquots at a
concentration of 1 ng/mL in serum-free RPMI 1640 medium (this
concentration being the result of titration experiments in which
LPS concentration was varied between 50 pg/mL and 100 ng/mL). This
incubation was performed in the absence or presence of rBPI.sub.21
or rBPI(10-193)C132A in varying concentrations between 25 ng/mL and
50 .mu.g/mL. Recombinant human rBPI.sub.21, also designated
rBPI.sub.21cys, which is rBPI 1-193 with alanine substituted at
position 132 for cysteine [see co-owned U.S. Pat. No. 5,420,019],
was used as a positive control at a concentration of 1 .mu.g/mL.
Cell proliferation was quantitatively measured by the addition of 1
.mu.Ci/well [.sup.3H]-thymidine 5 hours after the time of
initiation of the assay. After the 15-hour incubation, labeled
cells were harvested onto glass fiber filters with a cell harvester
(Inotech Biosystems, INB-384, Sample Processing and Filter Counting
System, Lansing, Mich.). The LPS-mediated inhibition of RAW 264.7
cell proliferation is dependent on the presence of LBP, as added to
the reaction mixture either as a component of serum or as
recombinant LBP (at a concentration of 1 .mu.g/mL.
[0074] In these experiments, both rBPI.sub.21 and rBPI(10-193)C132A
similarly inhibited the LPS-mediated inhibition of RAW cell
proliferation.
[0075] B. Activity in a TNF Inhibition Assay
[0076] A tumor necrosis factor (TNF) inhibition assay was used to
compare the in vitro LPS neutralization activity of rBPI.sub.21 and
the rBPI(10-193)C132A. In this assay, the LPS, in combination with
purified LBP (or serum containing LBP) stimulates synthesis of TNF
by THP-1 cells (a human monocyte cell line), and rBPI.sub.21
neutralizes this effect of LPS.
[0077] THP.1 cells (ATCC Accession No. TIB-202) were maintained in
RPMI (GibcoBRL, Gaithersburg, Md.) with 10% FBS and were cultured
in RPMI with 10% FBS plus 50 ng/ml 1,25 dihydroxy vitamin D (BIOMOL
Research Laboratories Inc. Plymouth Meeting, Pa.) for three days
prior to treatment with LPS to induce CD14 expression. Before
inducing with LPS, cells were washed three times with RPMI and
suspended in either RPMI with 10% FBS or in serum free medium [RPMI
supplemented with 1% HB101 (Irvine Scientific, Santa Ana, Calif.)].
Expression of TNF was induced with 1 ng/ml E. coli 0128 LPS (Sigma,
St. Louis, Mo.) in 96 well plates with approximately
5.times.10.sup.4 cells per well. Plates were incubated for three
hours at 37.degree. C., 5% CO.sub.2, then an aliquot of the
supernatant liquid was removed and assayed for TNF by the WEHI 164
toxicity assay, u sing CellTiter 96.TM. A Q (Promega Corp.,
Madison, Wis.) to monitor c ell viability. Recombinant human
TNF.alpha. (Genzyme Diagnostics, Cambridge, Mass.) was used as a
positive standard. Both rBPI.sub.21 and rBPI(10-193)C132A similarly
inhibited LPS-induced stimulation of TNF synthesis.
EXAMPLE 8
In Vivo Biological Activity of rBPI(10-193)C132A
[0078] The in vivo assays described below were performed using the
purified rBPI(10-193)C132A produced in the 500-liter fermenter
according to Example 3B.
[0079] A. Toxicity Study in Rats
[0080] Toxicity profiles of rBPI.sub.21 and rBPI(10-193)C132A were
compared in rats. In this study, groups of six male and six female
Sprague-Dawley rats received either vehicle control (formulation
buffer), low (50 mg/kg/day) or high (120 mg/kg/day) doses of either
rBPI.sub.21 or rBPI(10-193)C132A. Doses were administered by
continuous intravenous infusion via an indwelling femoral catheter
for three consecutive days at an infusion rate of 4.2 mL/kg/hour
(100 mL/kg/day). Clinical observations were recorded at least twice
daily and body weights were recorded daily. Blood and urine samples
were collected near termination for hematology, clinical chemistry
and urinalysis assessments. At termination, organs were weighed and
tissues collected by histopathological examination. There were no
deaths or significant test article-related effects. The data
indicated similar toxicity profiles for rBPI.sub.21 and the
rBPI(10-193)C132A when given by continuous infusion.
[0081] B. Pharmacokinetics
[0082] The pharmacokinetics of rBPI.sub.21 and rBPI(10-193)C132A at
2 mg/kg were investigated in rats. The plasma clearances of
rBPI.sub.21 and rBPI(10-193)C132A were well described by a
tri-exponential pharmacokinetic disposition function. No
statistical differences in the pharmacokinetic parameters among the
rBPI products were determined (non parametric Wicoxon rank test,
p<0.05). Most of the administered drug (>96%) was cleared
with an alpha phase half-life of 0.2-0.4 minutes and a beta
half-life of 3.9-4.3 minutes, while the remainder was cleared
during the gamma phase with a half-life of 27-33 minutes. The
volume of distribution of the central compartment (Vc) was 41-45
mL/kg, and the clearance rate (CL) was 24-30 mL/min/kg. The steady
state volume of distribution was 152-184 mL/kg.
[0083] C. Efficacy In Mouse Endotoxin Challenge
[0084] Two separate studies were conducted to examine relative
potencies of rBPI.sub.21 and rBPI(10-193)C132A in a mouse model of
lethal endotoxemia generally according to Ammons et al., in "Novel
Therapeutic Strategies in the Treatment of Sepsis," Morrison and
Ryan, eds., Marcel Dekker, New York (1996), pages 55-69. In both
studies, there were 14 mice in each treatment and control group. In
the first study, CD1 mice were challenged intravenously with 25
mg/kg of lipopolysaccharide (LPS) from E. coli O111:B4. Immediately
after the challenge, the mice were treated intravenously with
rBPI.sub.21 or rBPI(10-193)C132A at doses of 15, 20, 25 and 30
mg/kg, or with the control vehicle (formulation buffer only).
Mortality was recorded twice daily for seven days.
[0085] The results from the first study, shown in Table 1 below,
indicate that treatment with both rBPI(10-193)C132A and rBPI.sub.21
significantly increased survival compared to the vehicle controls.
In addition, rBPI(10-193)C132A was at least two-fold more potent
than rBPI.sub.21 with a similar survival benefit seen with a
two-fold lower dose of rBPI(10-193)C132A compared to
rBPI.sub.21.
2 TABLE 1 No. of Survivors out of 15 Dose (mg/kg) Control
rBPI.sub.21 rBPI(10-193)C132A 0(Vehicle) 0 NA.sup.1 NA 15 0 15**.##
20 4 15**,# 25 11** 15** 30 13** 13** .sup.1NA, Not Applicable **,
p < 0.01 vs. control #, p < 0.05 vs. rBPI.sub.21 ##, p <
0.01 vs. control
[0086] In the second study, a wider range of rBPI(10-193)C132A
doses (5, 10, 15, 20, 25, 30 mg/kg) was studied. The results, shown
in Table 2 below, confirm that while both rBPI.sub.21 and
rBPI(10-193)C132A offered a significant survival benefit over the
control, as in the first study, rBPI(10-193)C132A was at least
two-fold more potent, achieving similar efficacy as rBPI.sub.21
with a 2-fold lower dose.
3 TABLE 2 No. of Survivors out of 15 rBPI(10- Dose (mg/kg) Control
rBPI.sub.21 193)C132A 0(Vehicle) 2 NA.sup.1 NA 5 ND.sup.1 3 10 ND
10* 15 ND 14** 20 7 14**,# 25 13** 15** 30 14** 15* .sup.1NA, Not
Applicable; ND, Not Done **, p < 0.05 vs. control **, p <
0.01 vs. control #, p < 0.05 vs. rBPI.sub.21
[0087] D. Efficacy in Murine Model of Lethal Bacteremia
[0088] Two separate studies were conducted to examine the relative
potencies or rBPI.sub.21 and rBPI(10-193)C132A in a mouse model of
lethal bacteremia. In both studies, t here w ere 20 mice p er
treatment group. In the first study, C D1 m ice were challenged
with 6.8.times.10.sup.7 colony forming units (CFU) of E. coli 07:K1
administered intravenously. Immediately after the challenge, the
mice were treated intravenously with rBPI.sub.21 or
rBPI(10-193)C132A at doses of 10, 20 and 30 mg/kg, or with control
vehicle (formulation buffer only). Mortality was recorded twice
daily for seven days.
[0089] The results from the first study, shown in Table 3 below,
demonstrate a significant increase in survival for the groups
treated with 10 and 30 m g/kg o f rBPI.sub.21 (p<0.05 vs.
control). While a similar significant increase in survival was not
observed with the rBPI(10-193)C132A vs. control, there was not a
significant difference in survival advantage between the
rBPI.sub.21 and rBPI(10-193)C132A-treated groups in this study.
4 TABLE 3 No. of Survivors out of 20 Dose (mg/kg) Control
rBPI.sub.21 rBPI(10-193)C132A 0(Vehicle) 6 NA 10 14* 12 20 12 10 30
14* 10 *, p < 0.05 vs. control
[0090] To more fully characterize the effects of rBPI.sub.21 and
rBPI(10-193)C132A in this model, a second study was conducted in
which a wider range of doses was studied. In this study, CD1 mice
were challenged with 2.57.times.10.sup.8 colony forming units (CFU)
of E. Coli 07:K1 administered intravenously. Immediately after the
challenge, the mice were treated intravenously with 1.0, 3.0, 10
and 30 mg/kg rBPI.sub.21 and 0.3, 1.0, 3.0, 10 and 30 mg/kg
rBPI(10-193)C132A. The results, shown in Table 4 below, indicate
that both proteins provided protection, and that there was no
significant difference in the protective effects of the two
variants at any dose.
5 TABLE 4 No. of Survivors out of 20 Dose (mg/kg) Control
rBPI.sub.21 rBPI(10-193)C132A 0(Vehicle) 6 0.3 ND.sup.1 6 1.0 4 6
3.0 9 10* 10 13** 8 30 11* 14** .sup.1ND, Not Done *, p < 0.05
vs. control **, p < 0.01 vs. control
[0091] E. Cardiovascular Effects in Conscious Rats
[0092] A series of experiments were conducted to determine the
relative effects of rBPI.sub.21 and rBPI(10-193)C132A on blood
pressure in rats. Each rat was anesthetized with a mixture of
ketamine (Fort Dodge Labs, Fort Dodge, Iowa) and Rompum (Bayer
Corp., Shawnee Mission, Kans.). A catheter was then placed in the
right carotid artery and connected to a pressure transducer to
record blood pressure. A second catheter was placed in the right
jugular vein to inject rBPI or vehicle. The rats were then allowed
to recover before the experiments began. Experiments were initiated
when the rats were alert, mobile and when blood pressure was stable
within the normal range. rBPI.sub.21, rBPI(10-193)C132A or control
vehicle (formulation buffer) were then injected as a bolus over 15
seconds and mean arterial blood pressure (mm Hg) was recorded over
the next 60 minutes.
[0093] In preliminary experiments, it was determined that doses of
20 and 30 mg/kg of rBPI.sub.21 had no significant effect on blood
pressure relative to the vehicle but that a dose of 40 mg/kg
resulted in a significant decrease in blood pressure that was
evident within 5 minutes. This hypotensive response was greatest 15
minutes after the injection when blood pressure had decreased by
48.+-.12 mm Hg (mean.+-.SE; p>0.05). After 60 minutes, the blood
pressure of the rBPI.sub.21-treated animals recovered and was not
significantly different from that of the vehicle-treated
animals.
[0094] To compare effects of rBPI.sub.21 and rBPI(10-193)C132A,
groups of 5 rats were given 40 mg/kg of each drug substance or
vehicle control, and blood pressure responses were analyzed as area
under the curve (AUC). FIG. 1 shows that, as previously observed,
rBPI.sub.21 caused a significant drop in blood pressure indicated
by the elevated AUC relative to the vehicle control. By comparison,
rBPI(10-193)C132A had no significant effect on blood pressure
compared with the vehicle control. A dose of 50 mg/kg rBPI.sub.21
(N=4 rats) had an even greater hypotensive effect than that of the
40 mg/kg dose as indicated by a further increase in the AUC in FIG.
1. At this higher dose, some reduction in blood pressure also
occurred in rats administered rBPI(10-193)C132A (N=3), but this
effect was not significant compared to the vehicle control.
[0095] Numerous modifications and variations of the above-described
invention are expected to occur to those of skill in the art.
Accordingly, only such limitations as appear in the appended claims
should be placed thereon.
Sequence CWU 1
1
6 1 1813 DNA Homo sapiens CDS (31)..(1491) mat_peptide
(124)..(1491) rBPI 1 caggccttga ggttttggca gctctggagg atg aga gag
aac atg gcc agg ggc 54 Met Arg Glu Asn Met Ala Arg Gly -30 -25 cct
tgc aac gcg ccg aga tgg gtg tcc ctg atg gtg ctc gtc gcc ata 102 Pro
Cys Asn Ala Pro Arg Trp Val Ser Leu Met Val Leu Val Ala Ile -20 -15
-10 ggc acc gcc gtg aca gcg gcc gtc aac cct ggc gtc gtg gtc agg atc
150 Gly Thr Ala Val Thr Ala Ala Val Asn Pro Gly Val Val Val Arg Ile
-5 -1 1 5 tcc cag aag ggc ctg gac tac gcc agc cag cag ggg acg gcc
gct ctg 198 Ser Gln Lys Gly Leu Asp Tyr Ala Ser Gln Gln Gly Thr Ala
Ala Leu 10 15 20 25 cag aag gag ctg aag agg atc aag att cct gac tac
tca gac agc ttt 246 Gln Lys Glu Leu Lys Arg Ile Lys Ile Pro Asp Tyr
Ser Asp Ser Phe 30 35 40 aag atc aag cat ctt ggg aag ggg cat tat
agc ttc tac agc atg gac 294 Lys Ile Lys His Leu Gly Lys Gly His Tyr
Ser Phe Tyr Ser Met Asp 45 50 55 atc cgt gaa ttc cag ctt ccc agt
tcc cag ata agc atg gtg ccc aat 342 Ile Arg Glu Phe Gln Leu Pro Ser
Ser Gln Ile Ser Met Val Pro Asn 60 65 70 gtg ggc ctt aag ttc tcc
atc agc aac gcc aat atc aag atc agc ggg 390 Val Gly Leu Lys Phe Ser
Ile Ser Asn Ala Asn Ile Lys Ile Ser Gly 75 80 85 aaa tgg aag gca
caa aag aga ttc tta aaa atg agc ggc aat ttt gac 438 Lys Trp Lys Ala
Gln Lys Arg Phe Leu Lys Met Ser Gly Asn Phe Asp 90 95 100 105 ctg
agc ata gaa ggc atg tcc att tcg gct gat ctg aag ctg ggc agt 486 Leu
Ser Ile Glu Gly Met Ser Ile Ser Ala Asp Leu Lys Leu Gly Ser 110 115
120 aac ccc acg tca ggc aag ccc acc atc acc tgc tcc agc tgc agc agc
534 Asn Pro Thr Ser Gly Lys Pro Thr Ile Thr Cys Ser Ser Cys Ser Ser
125 130 135 cac atc aac agt gtc cac gtg cac atc tca aag agc aaa gtc
ggg tgg 582 His Ile Asn Ser Val His Val His Ile Ser Lys Ser Lys Val
Gly Trp 140 145 150 ctg atc caa ctc ttc cac aaa aaa att gag tct gcg
ctt cga aac aag 630 Leu Ile Gln Leu Phe His Lys Lys Ile Glu Ser Ala
Leu Arg Asn Lys 155 160 165 atg aac agc cag gtc tgc gag aaa gtg acc
aat tct gta tcc tcc aag 678 Met Asn Ser Gln Val Cys Glu Lys Val Thr
Asn Ser Val Ser Ser Lys 170 175 180 185 ctg caa cct tat ttc cag act
ctg cca gta atg acc aaa ata gat tct 726 Leu Gln Pro Tyr Phe Gln Thr
Leu Pro Val Met Thr Lys Ile Asp Ser 190 195 200 gtg gct gga atc aac
tat ggt ctg gtg gca cct cca gca acc acg gct 774 Val Ala Gly Ile Asn
Tyr Gly Leu Val Ala Pro Pro Ala Thr Thr Ala 205 210 215 gag acc ctg
gat gta cag atg aag ggg gag ttt tac agt gag aac cac 822 Glu Thr Leu
Asp Val Gln Met Lys Gly Glu Phe Tyr Ser Glu Asn His 220 225 230 cac
aat cca cct ccc ttt gct cca cca gtg atg gag ttt ccc gct gcc 870 His
Asn Pro Pro Pro Phe Ala Pro Pro Val Met Glu Phe Pro Ala Ala 235 240
245 cat gac cgc atg gta tac ctg ggc ctc tca gac tac ttc ttc aac aca
918 His Asp Arg Met Val Tyr Leu Gly Leu Ser Asp Tyr Phe Phe Asn Thr
250 255 260 265 gcc ggg ctt gta tac caa gag gct ggg gtc ttg aag atg
acc ctt aga 966 Ala Gly Leu Val Tyr Gln Glu Ala Gly Val Leu Lys Met
Thr Leu Arg 270 275 280 gat gac atg att cca aag gag tcc aaa ttt cga
ctg aca acc aag ttc 1014 Asp Asp Met Ile Pro Lys Glu Ser Lys Phe
Arg Leu Thr Thr Lys Phe 285 290 295 ttt gga acc ttc cta cct gag gtg
gcc aag aag ttt ccc aac atg aag 1062 Phe Gly Thr Phe Leu Pro Glu
Val Ala Lys Lys Phe Pro Asn Met Lys 300 305 310 ata cag atc cat gtc
tca gcc tcc acc ccg cca cac ctg tct gtg cag 1110 Ile Gln Ile His
Val Ser Ala Ser Thr Pro Pro His Leu Ser Val Gln 315 320 325 ccc acc
ggc ctt acc ttc tac cct gcc gtg gat gtc cag gcc ttt gcc 1158 Pro
Thr Gly Leu Thr Phe Tyr Pro Ala Val Asp Val Gln Ala Phe Ala 330 335
340 345 gtc ctc ccc aac tcc tcc ctg gct tcc ctc ttc ctg att ggc atg
cac 1206 Val Leu Pro Asn Ser Ser Leu Ala Ser Leu Phe Leu Ile Gly
Met His 350 355 360 aca act ggt tcc atg gag gtc agc gcc gag tcc aac
agg ctt gtt gga 1254 Thr Thr Gly Ser Met Glu Val Ser Ala Glu Ser
Asn Arg Leu Val Gly 365 370 375 gag ctc aag ctg gat agg ctg ctc ctg
gaa ctg aag cac tca aat att 1302 Glu Leu Lys Leu Asp Arg Leu Leu
Leu Glu Leu Lys His Ser Asn Ile 380 385 390 ggc ccc ttc ccg gtt gaa
ttg ctg cag gat atc atg aac tac att gta 1350 Gly Pro Phe Pro Val
Glu Leu Leu Gln Asp Ile Met Asn Tyr Ile Val 395 400 405 ccc att ctt
gtg ctg ccc agg gtt aac gag aaa cta cag aaa ggc ttc 1398 Pro Ile
Leu Val Leu Pro Arg Val Asn Glu Lys Leu Gln Lys Gly Phe 410 415 420
425 cct ctc ccg acg ccg gcc aga gtc cag ctc tac aac gta gtg ctt cag
1446 Pro Leu Pro Thr Pro Ala Arg Val Gln Leu Tyr Asn Val Val Leu
Gln 430 435 440 cct cac cag aac ttc ctg ctg ttc ggt gca gac gtt gtc
tat aaa 1491 Pro His Gln Asn Phe Leu Leu Phe Gly Ala Asp Val Val
Tyr Lys 445 450 455 tgaaggcacc aggggtgccg ggggctgtca gccgcacctg
ttcctgatgg gctgtggggc 1551 accggctgcc tttccccagg gaatcctctc
cagatcttaa ccaagagccc cttgcaaact 1611 tcttcgactc agattcagaa
atgatctaaa cacgaggaaa cattattcat tggaaaagtg 1671 catggtgtgt
attttaggga ttatgagctt ctttcaaggg ctaaggctgc agagatattt 1731
cctccaggaa tcgtgtttca attgtaacca agaaatttcc atttgtgctt catgaaaaaa
1791 aacttctggt ttttttcatg tg 1813 2 487 PRT Homo sapiens 2 Met Arg
Glu Asn Met Ala Arg Gly Pro Cys Asn Ala Pro Arg Trp Val -30 -25 -20
Ser Leu Met Val Leu Val Ala Ile Gly Thr Ala Val Thr Ala Ala Val -15
-10 -5 -1 1 Asn Pro Gly Val Val Val Arg Ile Ser Gln Lys Gly Leu Asp
Tyr Ala 5 10 15 Ser Gln Gln Gly Thr Ala Ala Leu Gln Lys Glu Leu Lys
Arg Ile Lys 20 25 30 Ile Pro Asp Tyr Ser Asp Ser Phe Lys Ile Lys
His Leu Gly Lys Gly 35 40 45 His Tyr Ser Phe Tyr Ser Met Asp Ile
Arg Glu Phe Gln Leu Pro Ser 50 55 60 65 Ser Gln Ile Ser Met Val Pro
Asn Val Gly Leu Lys Phe Ser Ile Ser 70 75 80 Asn Ala Asn Ile Lys
Ile Ser Gly Lys Trp Lys Ala Gln Lys Arg Phe 85 90 95 Leu Lys Met
Ser Gly Asn Phe Asp Leu Ser Ile Glu Gly Met Ser Ile 100 105 110 Ser
Ala Asp Leu Lys Leu Gly Ser Asn Pro Thr Ser Gly Lys Pro Thr 115 120
125 Ile Thr Cys Ser Ser Cys Ser Ser His Ile Asn Ser Val His Val His
130 135 140 145 Ile Ser Lys Ser Lys Val Gly Trp Leu Ile Gln Leu Phe
His Lys Lys 150 155 160 Ile Glu Ser Ala Leu Arg Asn Lys Met Asn Ser
Gln Val Cys Glu Lys 165 170 175 Val Thr Asn Ser Val Ser Ser Lys Leu
Gln Pro Tyr Phe Gln Thr Leu 180 185 190 Pro Val Met Thr Lys Ile Asp
Ser Val Ala Gly Ile Asn Tyr Gly Leu 195 200 205 Val Ala Pro Pro Ala
Thr Thr Ala Glu Thr Leu Asp Val Gln Met Lys 210 215 220 225 Gly Glu
Phe Tyr Ser Glu Asn His His Asn Pro Pro Pro Phe Ala Pro 230 235 240
Pro Val Met Glu Phe Pro Ala Ala His Asp Arg Met Val Tyr Leu Gly 245
250 255 Leu Ser Asp Tyr Phe Phe Asn Thr Ala Gly Leu Val Tyr Gln Glu
Ala 260 265 270 Gly Val Leu Lys Met Thr Leu Arg Asp Asp Met Ile Pro
Lys Glu Ser 275 280 285 Lys Phe Arg Leu Thr Thr Lys Phe Phe Gly Thr
Phe Leu Pro Glu Val 290 295 300 305 Ala Lys Lys Phe Pro Asn Met Lys
Ile Gln Ile His Val Ser Ala Ser 310 315 320 Thr Pro Pro His Leu Ser
Val Gln Pro Thr Gly Leu Thr Phe Tyr Pro 325 330 335 Ala Val Asp Val
Gln Ala Phe Ala Val Leu Pro Asn Ser Ser Leu Ala 340 345 350 Ser Leu
Phe Leu Ile Gly Met His Thr Thr Gly Ser Met Glu Val Ser 355 360 365
Ala Glu Ser Asn Arg Leu Val Gly Glu Leu Lys Leu Asp Arg Leu Leu 370
375 380 385 Leu Glu Leu Lys His Ser Asn Ile Gly Pro Phe Pro Val Glu
Leu Leu 390 395 400 Gln Asp Ile Met Asn Tyr Ile Val Pro Ile Leu Val
Leu Pro Arg Val 405 410 415 Asn Glu Lys Leu Gln Lys Gly Phe Pro Leu
Pro Thr Pro Ala Arg Val 420 425 430 Gln Leu Tyr Asn Val Val Leu Gln
Pro His Gln Asn Phe Leu Leu Phe 435 440 445 Gly Ala Asp Val Val Tyr
Lys 450 455 3 21 DNA Artificial Sequence Description of Artificial
Sequence primer 3 ctgctctaaa agctgctgca g 21 4 33 DNA Artificial
Sequence Description of Artificial Sequence primer 4 ccaggccctt
ctgggaggcc gctgtcacgg cgg 33 5 33 DNA Artificial Sequence
Description of Artificial Sequence primer 5 gccgtgacag cggcctccca
gaagggcctg gac 33 6 18 DNA Artificial Sequence Description of
Artificial Sequence primer 6 ctgggaactg ggaagctg 18
* * * * *