U.S. patent application number 10/980722 was filed with the patent office on 2006-01-12 for stable bactericidal/permeability-increasing protein products and pharmaceutical compositions containing the same.
Invention is credited to Manik Baltaian, David Burke, Lynn Grinna, Arnold Horwitz, Georgia Theofan.
Application Number | 20060009383 10/980722 |
Document ID | / |
Family ID | 21761817 |
Filed Date | 2006-01-12 |
United States Patent
Application |
20060009383 |
Kind Code |
A1 |
Theofan; Georgia ; et
al. |
January 12, 2006 |
Stable bactericidal/permeability-increasing protein products and
pharmaceutical compositions containing the same
Abstract
Disclosed are novel bactericidal/permeability-increasing (BPI)
protein products wherein cysteine residue number 132 or 135 is
replaced by another amino acid residue, preferably an alanine or
serine residue and/or wherein the leucine residue at position 193
is the carboxy terminal residue. Also disclosed are DNA sequences
encoding methods for the production of the same in appropriate host
cells, and stable homogeneous pharmaceutical compositions
containing the analogs suitable for use treatment of gram negative
bacterial infection and its sequelae.
Inventors: |
Theofan; Georgia; (Torrance,
CA) ; Horwitz; Arnold; (Los Angeles, CA) ;
Burke; David; (Oakland, CA) ; Baltaian; Manik;
(Glendale, CA) ; Grinna; Lynn; (Middleburg,
VA) |
Correspondence
Address: |
MARSHALL, GERSTEIN & BORUN LLP
233 S. WACKER DRIVE, SUITE 6300
SEARS TOWER
CHICAGO
IL
60606
US
|
Family ID: |
21761817 |
Appl. No.: |
10/980722 |
Filed: |
November 3, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10196460 |
Jul 16, 2002 |
6828418 |
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10980722 |
Nov 3, 2004 |
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09425034 |
Oct 19, 1999 |
6433140 |
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10196460 |
Jul 16, 2002 |
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09176493 |
Oct 21, 1998 |
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09425034 |
Oct 19, 1999 |
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08466822 |
Jun 6, 1995 |
5827816 |
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09176493 |
Oct 21, 1998 |
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08361191 |
Apr 18, 1995 |
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08466822 |
Jun 6, 1995 |
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08013801 |
Feb 2, 1993 |
5420019 |
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08361191 |
Apr 18, 1995 |
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Current U.S.
Class: |
514/2.2 ;
514/2.8; 530/350 |
Current CPC
Class: |
A61P 31/04 20180101;
A61P 11/00 20180101; A61P 29/00 20180101; A61K 38/00 20130101; A61P
7/06 20180101; C12N 2830/30 20130101; A61P 7/02 20180101; A61P 9/02
20180101; A61P 31/00 20180101; C12N 2830/00 20130101; C12N 2840/44
20130101; A61P 13/12 20180101; C12N 2830/60 20130101; A61P 39/00
20180101; C12N 2830/85 20130101; C07K 19/00 20130101; A61P 39/02
20180101; C07K 2319/00 20130101; A61P 7/00 20180101; C12N 2830/15
20130101; C12N 15/85 20130101; C07K 14/4742 20130101 |
Class at
Publication: |
514/012 ;
530/350 |
International
Class: |
A61K 38/16 20060101
A61K038/16; C07K 14/195 20060101 C07K014/195 |
Claims
1. A polypeptide analog of bactericidal/permeability-increasing
protein or biologically-active fragment thereof wherein a cysteine
residue at position 132 or at position 135 is replaced by a
different amino acid.
2. The polypeptide 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 polypeptide analog of claim 1 wherein the cysteine residue
at position 132 is replaced by alanine.
4. The polypeptide analog of claim 1 wherein the cysteine residue
at position 135 is replaced by serine.
5. The polypeptide analog according to claims 1, 2, 3, or 4
comprising, except for the cysteine replacement at either position
132 or 135, from 176 to 199 of the amino terminal residues of
bactericidal/permeability-increasing protein.
6. The polypeptide analog according to claim 5 comprising the
initial 193 amino terminal amino acid residues of
bactericidal/permeability-increasing protein.
7. A DNA encoding a polypeptide analog of
bactericidal/permeability-increasing protein or a biologically
active fragment thereof wherein a cysteine residue at position 132
or at position 135 is replaced by a different amino acid.
8. The DNA of claim 7 encoding the thirty-one amino acid leader
sequence and the first 193 N-terminal residues of
bacterial/permeability-increasing protein and having a stop codon
immediately following the codon for the leucine residue at position
193:
9. The DNA of claim 7 encoding the thirty-one amino acid leader
sequence and the first 199 N-terminal residues of
bactericidal/permeability-increasing protein and having a stop
codon immediately following the codon for the isoleucine at
position 199.
10. An autonomously replicating DNA vector comprising a DNA
according to claim 7.
11. A host cell stably transformed or transfected with DNA
according to claim 7 in a manner allowing expression in said host
cell of said polypeptide analog.
12. A eukaryotic host cell according to claim 11.
13. A host cell according to claim 12 selected from the group
consisting of ATCC CRL 11246 and ATCC HB11247.
14. A method for producing polypeptide analogs of
bactericidal/permeability increasing protein and biologically
active fragments thereof comprising growing a host cell according
to claim 11 in a suitable culture medium and isolating said analog
from said host cell or said culture medium.
15. A hybrid fusion protein comprising, at its amino terminal, an
analog polypeptide according to claim 1 and, at its carboxyl
terminal, at least one constant domain of an immunoglobulin heavy
chain or an allelic variation thereof.
16. A DNA encoding a hybrid fusion protein according to claim
15.
17. An autonomously replicating DNA vector comprising a DNA
according to claim 16.
18. A host cell stably transformed or transfected with a DNA
according claim 16 in a manner allowing expression in said host
cell of said fusion polypeptide.
19. A method for producing hybrid fusion protein according to claim
15 comprising growing a host cell according to claim 18 in a
suitable culture medium and isolating said analog from said host
cell or said culture medium.
20. A pharmaceutical composition comprising an analog polypeptide
according to claim 1 and a pharmaceutically-acceptable diluent,
adjuvant, or carrier.
21. A pharmaceutical composition comprising a hybrid fusion protein
according to claim 15 and a pharmaceutically acceptable diluent
adjuvant, or carrier.
22. A method of treating bacterial infection or the sequelae
thereof comprising administering an effective amount of the
pharmaceutical composition of claims 20 or 21.
23. A DNA encoding a biologically active fragment of
bactericidal/permeability increasing protein having a leucine
residue at position 193 as its carboxy terminal residue.
24. The DNA according to claim 23 encoding the thirty-one amino
acid leader sequence and first 193 N-terminal amino acids of
bacterial/permeability-increasing protein and having a stop codon
immediately following the codon for the leucine residue at position
193.
25. An autonomously replicating DNA vector comprising a DNA
according to claim 22.
26. A host cell stably transformed or transfected with a DNA
according to claim 23 in manner allowing expression in said host
cell.
27. A method for producing a biologically active bactericid
al/permeability increasing protein fragment comprising growing a
host cell stably transformed or transfected with DNA according to
claim 23 in a suitable culture medium and isolating said fragment
from said host cell or said culture medium.
Description
BACKGROUND OF THE INVENTION
[0001] The present invention provides novel
bactericidal/permeability-increasing protein products and stable
pharmaceutical compositions containing the same.
[0002] Lipopolysaccharide (LPS), is a major component of the outer
membrane of gram-negative bacteria and consists of
serotype-specific O-side-chain polysaccharides linked to a
conserved region of core oligosaccharide and lipid A. Raetz, Ann.
Rev. Biochem., 59:129-170 (1990). LPS is an important mediator in
the pathogenesis of gram-negative septic shock, one of the major
causes of death in intensive-care units in the United States.
Morrison, et al., Ann. Rev. Med. 38:417-432 (1987).
[0003] LPS-binding proteins have been identified in various
mammalian tissues. Morrison, Microb. Pathol., 7:389-398 (1989);
Roeder, et al., Infect., Immun., 57:1054-1058 (1989). Among the
most extensively studied of the LPS-binding proteins is
bactericidal/permeability-increasing protein (BPI), a basic protein
found in the azurophilic granules of polymorphonuclear leukocytes.
Human BPI protein has been isolated from polymorphonuclear
neutrophils 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)]
and has potent bactericidal activity against a broad spectrum of
gram-negative bacteria.
[0004] While the BPI protein is cytotoxic against many
gram-negative bacteria, it has no reported cytotoxic activity
toward gram-positive bacteria, fungi, or mammalian cells. The amino
acid sequence of the entire human BPI protein, as well as the DNA
encoding the protein, have been elucidated in FIG. 1 of Gray, et
al., J. Biol. Chem., 264:9505 (1989), incorporated herein by
reference (SEQ ID NOs: 1 and 2). The Gray et al. publication
discloses the isolation of human BPI-encoding cDNA from a cDNA
library derived from DMSO-induced cells of the human promyelocytic
leukemia HL-60 cell line (ATTC CCL 240). Multiple PCR
amplifications of DNA from a freshly prepared cDNA library derived
from such DMSO-induced HL-60 cells have revealed the existence of
human BPI-encoding cDNAs wherein the codon specifying valine at
amino acid position 151 is either GTC (as set out in SEQ ID No: 1)
or GTG. Moreover, cDNA species employing GTG to specify valine at
position 151 have also been found to specify either lysine (AAG)
for the position 185 amino acid (as in SEQ ID Nos: 1 and 2) or a
glutamic acid residue (GAG) at that position.
[0005] A proteolytic fragment corresponding to the N-terminal
portion of human BPI holoprotein possesses the antibacterial
efficacy of the naturally-derived 55 kDa human BPI holoprotein. In
contrast to the N-terminal portion, the C-terminal region of the
isolated human BPI protein displays only slightly detectable
anti-bacterial activity. Ooi, et al., J. Exp. Med., 174:649 (1991).
A BPI N-terminal fragment, comprising approximately the first 199
amino acids of the human BPI holoprotein, has been produced by
recombinant means as a 23 kDa protein. Gazzano-Santoro et al.,
Infect. Immun. 60:4754-4761 (1992).
[0006] The projected clinical use of BPI products for treatment of
gram-negative sepsis in humans has prompted significant efforts to
produce large quantities of recombinant BPI (rBPI) products
suitable for incorporation into stable, homogeneous pharmaceutical
preparations. For example, co-owned, co-pending U.S. patent
application Ser. No. 07/885,501 by Grinna discloses novel methods
for the purification of recombinant BPI products expressed in and
secreted from genetically transformed mammalian host cells in
culture. Efficacy of the purification processes is therein
demonstrated in the context of products of transformed CHO cells
which express DNA encoding the 31 amino acid "leader" sequence of
human BPI and the initial 199 amino terminal residues of the mature
protein (i.e. corresponding to the amino acids -31 through 199 of
SEQ ID NO: 2). Co-owned, co-pending U.S. patent application Ser.
No. 07/885,911 by Theofan, et al. is directed to novel,
recombinant-produced BPI protein analog products resulting from the
expression of DNA encoding the BPI leader sequence and either 191
or 199 amino terminal residues of human BPI fused to DNA encoding a
constant region of an immunoglobulin heavy chain.
[0007] Efforts to produce pharmaceutical grade BPI products for
treatment of gram negative sepsis in humans have not yielded
uniformly satisfactory results. A principal reason for this is the
nature of the amino acid sequence of human BPI and the nature of
the recombinant host cell environment in which the products are
produced. As one example, biologically-active rBPI products
comprising the initial 199 residues of BPI [rBPI(1-199)] produced
as secretory products of transfected CHO host cells may be purified
in good yields. However, the isolated BPI products initially
include dimeric forms of BPI and cysteine adduct species. Moreover,
BPI products may be unstable upon storage at physiological
temperature and pH, resulting in the formation of additional
dimeric and adduct species. Such dimeric and adduct species, while
retaining biological activity, are not preferred for incorporation
into pharmaceutical preparations projected for human use. Dimer
formation and the formation of cysteine adducts are the probable
result of the fact that BPI includes three cysteine amino acid
residues, all of which are positioned within the biologically
active amino terminal region of BPI, i.e., at positions 132, 135
and 175. Formation of a single disulfide bond between two of the
three cysteines allows for dimer formation or formation of cysteine
adducts with the remaining free cysteine in the host cell cytoplasm
and/or the cell culture supernatant.
[0008] Even monomeric rBPI products display varying degrees of
microheterogeneity in terms of the number of carboxy terminal
residues persent in such products. For example, it is difficult to
detect full-length expression product in a medium containing host
cells transformed or transfected with DNA encoding rBPI(1-199).
Instead, the expression products obtained from such cells represent
an heterogeneous array of carboxy-terminal truncated species of the
rBPI N-terminal fragment. In fact, the expected full-length product
(1-199) is often not detected as being among the rBPI species
present in that heterogeneous array. Heterogeneity of the carboxy
terminal amino acid sequence of rBPI(1-199) products appears to
result from activity of carboxypeptidases in host cell cytoplasm
and/or culture supernatant.
[0009] An additional problem encountered in the preparation of
pharmaceutical-grade BPI products is the formation of macroscopic
particles which decrease the homogeneity of the product, as well as
decreasing its activity. A preferred pharmaceutical composition
containing rBPI products according to the invention comprises the
combination of a poloxamer (polyoxypropylene-polyoxyethylene block
copolymer) surfactant and a polysorbate (polyoxyethylene sorbitan
fatty acid ester) surfactant. Such combinations are taught in
co-owned, co-pending concurrently filed U.S. patent application
Ser. No. (Attorney docket no. 27129/31162) to have synergistic
effects in stabilizing pharmaceutically-active polypeptides against
particle formation. Most preferred is a composition in which the
rBPI product is present in a concentration of 1 mg/ml in citrate
buffered saline (0.02 M citrate, 0.15 M NaCl, pH 5.0) comprising
0.1% by weight of poloxamer 188 (Pluronic F-68, BASF Wyandotte,
Passippany, N.J.) and 0.002% by weight of polysorbate 80 (Tween 80,
ICI Americase Inc., Wilmington, Del.).
[0010] There continues to be a need in the art for improved rBPI
products suitable for incorporation into stable homogeneous
pharmaceutical preparations. Such products would ideally be
obtainable in large yield from transformed host cells, would retain
the bactericidal and LPS-binding biological activities of BPI, and
would be limited in their capacity to form dimeric species and
cysteine adducts, and would be characterized by limited variation
in carboxy termini.
SUMMARY OF THE INVENTION
[0011] The present invention provides novel, biologically-active,
recombinant-produced BPI ("rBPI") protein and protein fragment
products which are characterized by a resistance to dimerization
and cysteine adduct formation, making such products highly suitable
for pharmaceutical use. Also provided are rBPI products
characterized by decreased molecular heterogeneity at the carboxy
terminus. Novel DNA sequences encoding rBPI products and analog
products, plasmid vectors containing the DNA, host cells stably
transformed or transfected with the plasmids, recombinant
preparative methods, stable pharmaceutical compositions and
treatment methods are also provided by the invention.
[0012] According to one aspect of the present invention, rBPI
protein analogs are provided which comprise a BPI N-terminal
fragment wherein a cysteine at amino acid position 132 or 135 is
replaced by another amino acid, preferably a non-polar amino acid
such as serine or alanine. In a preferred embodiment of the
invention, the cysteine residue at position 132 of a polypeptide
comprising the first 199 N-terminal residues of BPI is replaced by
an alanine residue in a recombinant product designated
"rBPI(1-199)ala.sup.132". Also in a preferred embodiment of the
invention, the cysteine at position 135 of a BPI fragment
comprising the first 199 N-terminal BPI residues is replaced by a
serine, resulting in a recombinant product designated
"rBPI(1-199)ser.sup.135". Highly preferred is a recombinant product
designated "rBPI(1-193)ala.sup.132" which is characterized by a
decreased heterogeneity in terms of the identity of its carboxy
terminal residue. Also in a preferred embodiment of the invention,
a polypeptide is taught which comprises the first 193
amino-terminal residues of BPI and which has a stop codon
immediately following the candon for Leucine at position 193.
[0013] According to another aspect of the invention, DNA sequences
are provided which encode the above-described rBPI protein and
protein fragment products, including analog products. Such DNA
sequences may also encode the 31-residue BPI leader sequence and
the BPI polyadenylation signal. Also provided are
autonomously-replicating DNA plasmid vectors which include DNA
encoding the above-mentioned products and analogs as well as host
cells which are stably transformed or transfected with the DNA in a
manner sufficient to allow their expression. Transformed or
transfected host cells according to the invention are of manifest
utility in methods for the large-scale production of rBPI protein
products of the invention.
[0014] The invention also contemplates rBPI protein analog products
in the form of fusion proteins comprising, at the amino terminal,
rBPI protein analog products of the invention and, at the carboxy
terminal, a constant region of an immunoglobulin heavy chain or an
allelic variant thereof. Natural sequence BPI/immunoglobulin fusion
proteins are taught in the co-pending, co-owned U.S. patent
application Ser. No. 07/885,911 by Theofan, et al., the disclosures
of which are incorporated herein by reference. The invention
further contemplates methods for producing the aforementioned
fusion proteins.
[0015] Also within the scope of the present invention are DNA
sequences encoding biologically-active rBPI protein fragment
products having from about 176 to about 198 of the N-terminal amino
acids of BPI. These DNAs allow for production of BPI products in
eukaryotic host cells, such as CHO cells, wherein the products
display less heterogeneity in terms of the carboxy terminal
residues present. Presently preferred are DNAs encoding 193
N-terminal residues of BPI (e.g., DNAs encoding the thirty-one
amino acid leader sequence of BPI, the initial 193 N-terminal amino
acids, and one or more stop codons). Most preferred are such DNAs
which additionally encode proteins wherein the cysteine at either
position 132 or 135 is replaced (e.g., rBPI(1-193)ala.sup.132).
[0016] Finally, the present invention also provides stable,
homogeneous pharmaceutical compositions comprising the rBPI protein
products of the invention in pharmaceutically acceptable diluents,
adjuvants, and carriers. Such pharmaceutical compositions are
resistant to the formation of rBPI product particles. Such
compositions are useful in the treatment of gram-negative bacterial
infection and the sequelae thereof, including endotoxin-related
shock and one or more conditions associated therewith, such as
disseminated intravascular coagulation, anemia, thrombocytopenia,
leukopenia, adult respiratory distress syndrome, renal failure,
hypotension, fever, and metabolic acidosis.
[0017] 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
presently preferred embodiments thereof.
BRIEF DESCRIPTION OF THE DRAWING
[0018] FIG. 1 represents results of SDS-PAGE analysis of
rBPI(1-193) products.
[0019] FIG. 2 represents results of SDS-PAGE analysis of
rBPI(1-193) and rBPI(1-193).sup.132 products.
[0020] FIG. 3 depicts results of cation exchange HPLC analysis of
rBPI(1-199) products.
[0021] FIG. 4 shows results of cation exchange HPLC analysis of
rBPI(1-199)ala.sup.132 products.
[0022] FIG. 5 represents results of reverse phase HPLC run on
rBPI(1-199) products.
[0023] FIG. 6 represents results of reverse phase HPLC run on
rBPI(1-199)ala.sup.132 products.
[0024] FIG. 7 presents results of turbidity studies on
pharmaceutical compositions containing rBPI products with and
without poloxamer/polysorbate surfactant ingredients at pH 7.0 and
57.degree. C.
[0025] FIG. 8 depicts comparative binding of rBPI(1-199) and
rBPI(1-199)ala.sup.132 to LPS.
[0026] FIG. 9 reports the bactericidal activity of
rBPI(1-199)ala.sup.132 products.
DETAILED DESCRIPTION
[0027] The following detailed description relates to the
manufacture and properties of various rBPI product preparations
which comprise an amino acid substitution at a cysteine residue
and/or highly uniform carboxy termini. More specifically, Example 1
relates to an exemplary means by which base substitutions are
introduced in the nucleotide sequence encoding an exemplary
N-terminal fragment of the BPI protein and to the incorporation of
such mutated sequences into plasmid vectors. Example 2 addresses
the incorporation of vectors of Example 1 into appropriate host
cells and further describes the expression of recombinant BPI
protein polypeptide products of the invention. Example 3 relates to
construction of DNAs encoding cysteine replacement analog products
of the invention and the use thereof in in vitro
transcription/translation procedures. Example 4 relates to
properties of rBPI product polypeptides of the invention.
EXAMPLE 1
Construction Of Vectors Containing Bpi Cysteine Replacement
Analogs
A. Construction Of Plasmids pING4519 And pING4520
[0028] The expression vector, pING4503, was used as a source of DNA
encoding a recombinant expression product designated rBPI(1-199),
i.e., encoding a polypeptide having the 31-residue signal sequence
and the first 199 amino acids of the N-terminus of the mature human
BPI, as set out in SEQ ID NOs: 1 and 2 except that valine at
position 151 is specified by GTG rather than GTC and residue 185 is
glutamic acid (specified by GAG) rather than a lysine (specified by
AAG).
[0029] Plasmid pING4503 has been described in co-pending, co-owned
U.S. patent application Ser. No. 07/885,911 by Theofan, et al.
which is incorporated herein by reference with respect to the
background of the invention. Briefly, the construction of pING4503
is based on plasmid pING2237N which contains the mouse
immunoglobulin heavy chain enhancer element, the LTR
enhancer-promoter element from Abelson murine leukemia virus
(A-MuLv) DNA, the SV40 19S/16S splice junction at the 5' end of the
gene to be expressed, and the human genomic gamma- 1
polyadenylation site at the 3' end of the gene to be expressed.
Plasmid pING2237N also has a mouse dihydrofolate reductase (DHFR)
selectable marker. The DNA encoding rBPI(1-199), including 30 bp of
the natural 5' untranslated region and bases encoding the 31 amino
acid signal sequence, as well as 199 N-terminal amino acids of BPI,
is inserted between unique SalI and SstII restriction sites in
pING45O3.
[0030] Two vectors, pING4519 and pING4520, were constructed based
on pING4503 for expression of rBPI(1-199) cysteine replacement
analogs in which one of the three naturally-occurring cysteine
residues of BPI was replaced with another amino acid. A PvuII site
(CAGCTG) which occurs only once in the DNA encoding rBPI(1-199),
and which is located between cysteine 132 and cysteine 135, was
utilized in these constructions. Because several additional PvuII
sites exist in pING4503, it was first necessary to isolate the
SalI-SstII fragment which contained the insert encoding rBPI(1-199)
from pING4503 by digesting with SalI and SstII. The purified
SalI-SstII rBPI(1-199) insert was then digested with PvuII,
resulting in an approximately 529 bp SalI-PvuII fragment and an
approximately 209 bp PvuII-SstII fragment, each of which was
purified separately.
[0031] Plasmid pING4519 is identical to pING4503 except that
pING4519 contains a DNA insert encoding an rBPI(1-199) in which a
codon for alanine is substituted for the codon specifying the
native cysteine at position 132. As noted above, the recombinant
product resulting from host cell expression and secretory
processing of such an insert is referred to as
"rBPI(1-199)ala.sup.132". In order to generate pING4519, BPI DNA
sequences were PCR amplified from pING45O3 using the primers BPI-6:
AAGCTTGTCGACCAGGCCTTGAGGT (SEQ ID NO: 3), which incorporated a SalI
restriction site at the 5' end of the 30 bp BPI untranslated
region, and BPI-14: CTGGAGGCGGTGATGGTG (SEQ ID NO: 4), which
incorporated one half of the PvuII site and the base substitutions
necessary to code for alanine at position 132. PCR amplification
was accomplished using the GeneAmp PCR kit (Perkin Elmer Cetus,
Norwalk, Conn.) according to the manufacturer's instructions. The
resulting PCR fragment was digested with SalI, resulting in an
approximately 529 bp SalI-blunt fragment which was then used in a
three-piece ligation, together with the approximately 209 bp
PvuII-SstII fragment described above and the large fragment
resulting from SalI and SstII digestion of pING4503 to generate
pING4519.
[0032] Plasmid pING4520 is identical to pING4519 with the exception
that pING4520 contains a DNA insert encoding an rBPI(1-199) analog
in which a serine codon is substituted for the codon specifying the
native cysteine at position 135. As noted above, the recombinant
product resulting from host cell expression of such an insert is
designated "rBPI(1-199) ser.sup.135". In order to generate
pING4520, BPI DNA sequences were PCR amplified from pING4513, a
plasmid essentially similar to pING4503 except that the selection
marker is gpt instead of DHFR and the cDNA insert encodes the
signal sequence and full-length BPI (456 residues) instead of only
the rBPI(1-199) portion.
[0033] Amplification by PCR was accomplished using primer BPI-15:
CTCCAGCAGCCACATCAAC (SEQ ID NO: 5), wherein the 5' end incorporates
one half of a mutated PvuII site (wherein "CTG" is changed to
"CTC") and the base substitutions necessary to code for serine at
position 135; and primer BPI-7: GAACTTGGTTGTCAGTCG (SEQ ID NO: 6),
representing rBPI-encoding sequences located downstream of the
region encoding BPI residue 199. This PCR fragment was digested
with BstBI, which cuts downstream of the cysteine 135 mutagenesis
site, and the resulting approximately 100 bp blunt-BstBI fragment
was gel purified. A three piece ligation was then performed with
the 529 bp SalI-PvuII BPI restriction fragment described above, the
100 bp blunt-BstBI fragment, and a large fragment resulting from
BstBI-SalI digestion of pING4503, to generate pING4520.
B. Construction Of Plasmid pING4530
[0034] Another vector, pING4530, was constructed which contained
the alanine-for-cysteine replacement as in pING4519, but which
contained the gpt selectable marker (allowing for mycophenolic acid
resistance) instead of the DHFR marker carried over from pING4503
to pING4519. To construct pING4530, a 1629 bp SalI-DraIII
restriction fragment was isolated from pING4519. This fragment
included all of the rBPI(1-199)ala.sup.132 coding region as well as
an additional approximately 895 bp vector sequence at the 3' end of
the coding region. This fragment was ligated to the large
(approximately 7230 bp) DraIII-SalI vector fragment isolated from
pING4513 to generate pING4530.
C. Construction Of Plasmid pING4533
[0035] Plasmid pING4533 was constructed for expression of
rBPI(1-199)ala .sup.32, wherein the codon specifying the fifth
amino acid of the BPI signal sequence, methionine (ATG), at
position -27 was placed in the context of the consensus Kozak
translation initiation sequence GCCACCRCCATGG (SEQ ID NO: 7)
[Kozak, Nucl. Acid. Res., 15:8125 (1987)], and in which the DNA
sequence encoding the first 4 amino acids of the BPI signal were
removed. This was accomplished by PCR amplification of BPI
sequences from a plasmid containing the full length human BPI cDNA
[in pGEM-7zf(+)] using the PCR primer BPI-23:
ACTGTCGACGCCACCATGGCCAGGGGC (SEQ ID NO: 8), incorporating a SalI
restriction site and the nucleotides GCCACC in front of the ATG
(methionine) at position -27 of the BPI signal, and the primer
BPI-2: CCGCGGCTCGAGCTATATTTTGGTCAT (SEQ ID NO: 9), corresponding to
the 3' end of the rBPI(1- 199) coding sequence.
[0036] The approximately 700 bp PCR amplified DNA was digested with
SalI and EcoRI and the resulting 270 bp fragment, including
approximately the first one-third of the BPI(1-199) coding
sequence, was purified. This SalI-EcoRI fragment was ligated to 2
other fragments: (1) a 420 bp EcoRI-SstII fragment from pING4519,
encoding the remainder of BPI(1-199) wherein alanine replaces
cysteine at position 132; and (2) an approximately 8000 bp
SstII-SalI vector fragment from pING4502 (a vector essentially
similar to pING4503 except that it does not include the 30 bp 5'
untranslated sequence and has a gpt marker rather than DHFR), to
generate pING4533 which contains a gpt marker.
D. Construction Of Plasmids pING4221, pING4222, And pING4223
[0037] Vectors similar to pING4533 were constructed having an
insert which contained the optimized Kozak translation initiation
site corresponding to methionyl residue -27 of the signal sequence,
and an alanine-for-cysteine replacement at position 132. However,
the BPI fragment coding sequence terminated at residue 193 in these
constructions. As noted above, the recombinant product resulting
from host cell expression of this DNA is referred to as
"rBP1(1-193)ala.sup.132". Vectors containing these inserts were
made by first digesting pING4533 with SalI, which cuts at the 5'
end of the BPI DNA insert, and AlwNI, which leaves a three bp
3'-overhang at residue 192. The resulting approximately 700 bp
fragment was then purified. This fragment was re-ligated into the
large fragment resulting from pING4533 digestion with SstII-SalI,
along with two annealed complementary oligonucleotides, BPI-30:
CTGTAGCTCGAGCCGC (SEQ ID NO: 10) and BPI-31: GGCTCGAGCTACAGAGT (SEQ
ID NO: 11). This replaced the region between the AlwNI and SstII
sites with the codon for residue 193 (leucine), a stop codon, and
an XhoI restriction site 5' to the SstII site and resulted in
regeneration of both the AlwNI and the SstII sites and placement of
the stop codon, TAG, immediately after the codon (CTG) for amino
acid 193 (leucine). The resultant plasmid was designated pING4223
and had the gpt marker. Similar constructions were made exactly as
described for pING4223 except that different SstII-SalI vector
fragments were used to generate vectors with different selection
markers. For example, pING4221 is identical to pING4223 except that
it contains the his marker (conferring resistance to histidinol)
instead of gpt and pING4222 is identical to pING4223 except that it
contains the DHFR marker instead of gpt.
E. Construction Of Plasmids pING4537, pING4143, pING 4146,
pING4150, And pING4154
[0038] A series of vectors was constructed which contained an
insert encoding rBP1(1-193)ala.sup.132, the optimized Kozak
translation initiation site, and different selection markers
essentially identical to those described with respect to pING4221,
pING4222 and pING4223 except that the human genomic gamma-1 heavy
chain polyadenylation and transcription termination region at the
3' end of the SstII site was replaced with a human light chain
polyadenylation sequence followed by mouse light chain (kappa)
genomic transcription termination sequences. In collateral gene
expression studies, the light chain polyadenylation signal and
transcription termination region appeared to be responsible for
2.5-5 fold increases in BPI expression levels in Sp2/0 and CHO-K1
cells.
[0039] The aforementioned vectors were constructed by first
constructing pING4537, a vector similar to pING4533 which contains
the rBPI(1-199)ala.sup.132 insert. However, pING4537 includes the
human light chain polyadenylation sequences instead of the human
heavy chain sequence. The mouse kappa 3' sequences were obtained
from pING3170, an expression vector which encodes a human light
chain cDNA and includes a mouse genomic light chain 3'
transcription termination sequence. This was accomplished by
digesting with SstI, which cuts 35 bp upstream of the mouse light
chain stop codon, treating with T4 DNA polymerase to make the end
blunt, then cutting with BamHI, and purifying an approximately 1350
bp fragment which includes the mouse kappa 3' sequences. The
resulting fragment consists of approximately 250 bp of the 3'
portion of the human light chain constant region cDNA and the
polyadenylation signal followed by a BamHI linker as described in
the construct called .DELTA.8 in Lui et al., J. Immunol. 139: 3521,
(1987). The remainder of the approximately 1350 bp fragment
consists of a BglII-BamHI mouse kappa 3' genomic fragment [fragment
"D" of Xu et al., J. Biol. Chem. 261:3838, (1986)] which supplies
transcription termination sequences. This fragment was used in a
3-piece ligation with two fragments from pING4533: a 3044 bp
fragment which includes all of BPI insert and part of vector
obtained by digestion with SstII, T4 polymerase treatment, and NotI
digestion (which includes all of BPI insert and part of vector),
and an approximately 4574 bp BamHI-NotI fragment. The resulting
vector, pING4537, is identical to pING4533 with the exception of
the above-noted differences in the genomic 3' untranslated
region.
[0040] Additional vectors containing the kappa 3' untranslated
sequences were constructed using pING4537 as the source of the
kappa 3' fragment. The kappa 3' untranslated sequences were
isolated by digestion of pING4537 with XhoI (a unique site which
occurs immediately after the BPI stop codon) and BamHI. The
resulting approximately 1360 bp XhoI-BamHI fragment was used in a
series of 3-piece ligations to generate the following four vectors,
all of which have inserts encoding rBPI(1-193)ala.sup.132 and which
have the optimized Kozak translation initiation site at residue -27
of the signal: (1) pING4143 (gpt marker), obtained by ligating a
pING4223 4574 bp BamHI-NotI fragment (gpt marker), a pING4223
NotI-XhoI BPI insert-containing fragment of approximately 3019 bp,
and the pING4537 XhoI-BamHI fragment; (2) pING4146 (DHFR marker),
obtained by ligating a pING4222 approximately 4159 bp BamHI-NotI
fragment (DHFR marker), a pING4223 NotI-XhoI BPI insert-containing
fragment of approximately 3019 bp, and the pING4537 XhoI-BamHI
fragment; (3) pING4150 (his marker), obtained by ligating a
pING4221 his-containing approximately 4772 bp BamHI-NotI fragment,
a pING4222 NotI-XhoI BPI insert-containing fragment, and the
pING4537 XhoI-BamHI fragment; and (4) pING4154 (neo marker),
obtained by ligating a pING3174 neo-containing approximately 4042
bp BamHI-BsaI fragment, a pING4221 BsaI-XhoI BPI insert-containing
fragment of approximately 3883 bp and the pING4537 XhoI-BamHI
fragment. Plasmid pING3174 contains an insert encoding antibody
heavy chain DNA and has a neo marker. The neo gene and its flanking
sequences were obtained from the pSv2 neo plasmid reported by
Southern et al., J. Mol. Appl. Genet., 1:327 (1982).
F. Construction Of Plasmids pING4144 And pING4151
[0041] Two plasmids were constructed, pING4144 and pING4151, which
were identical to pING4143 and pING4150, respectively, except that
expression of rBPI coding sequences was under control of the human
cytomegalovirus (hCMV) immediate early enhancer/promoter instead of
the Abelson murine leukemia virus (A-MuLv) LTR promoter. Therefore,
both pING4144 and pING4151 contained the mutation of the cysteine
at position 132 to alanine, the optimized Kozak translation
initiation sequence, and the human light chain poly-A/mouse kappa
genomic transcription termination region. The region between
nucleotides 879 and 1708 of the original vectors (pING4143 and
pING4150) was replaced with a region of the hCMV enhancer/promoter
corresponding to nucleotides -598 through +174 as shown in FIG. 3
of Boshart et al., Cell 41:521 (1985), incorporated herein by
reference. To introduce the hCMV promoter region into BPI
expression vectors, plasmid pING4538 was first constructed by
replacing the approximately 1117 bp EcoRI-SalI/A-MuLv
promoter-containing fragment of pING4222 with an approximately 1054
bp EcoRI-SalI/hCMV promoter-containing fragment from plasmid
pING2250 which contains the hCMV promoter driving expression of an
antibody light chain insert. To construct pING4144, three fragments
were ligated together: (1) the approximately 2955 bp
rBPI(1-193)-containing NotI-XhoI fragment from pING4538; (2) the
approximately 1360 bp XhoI-BamHI fragment from pING4537; and (3)
the approximately 4770 bp BamHI-NotI fragment containing the his
gene from pING4221.
G. Construction Of Plasmids pING4145, pING4148 And pING4152
[0042] Plasmids pING4145, pING4148 and pING4152 were constructed
and were identical to pING4143, pING4146, and pING4150,
respectively, except that they contained the wild-type (natural
sequence) cysteine at position 132 instead of an alanine
substitution. Thus, all three contained the rBPI(1-193) insert, the
optimized Kozak translation initiation sequence and the human light
chain Poly A/mouse kappa genomic transcription termination region.
These three plasmids were constructed as follows. To construct
pING4145, three fragments were ligated together: (1) the
approximately 3000 bp NotI-XhoI BPI(1 -193) containing fragment
from pING4140 (pING4140 is identical to pING4221 except that it
contains the wild-type cysteine at Position 132); (2) the
approximately 1360 bp XhoI-BamHI fragment from pING4537; and (3)
the approximately 4570 bp BamHI-NotI fragment containing the gpt
gene from pING4223. To construct pING4148, three fragments were
ligated together: (1) the NotI-XhoI fragment from pING4140; (2) the
XhoI-BamHI fragment from pING4537; and (3) the approximately 4150
bp BamHI-NotI fragment containing the DHFR gene from pING4222. To
construct pING4152, three fragments were ligated together: (1) the
approximately 3000 bp NotI-XhoI fragment from pING4142 (pING4142 is
identical to pING4223 except that it contains the wild-type
cysteine at 132); (2) the XhoI-BamHI fragment from pING4537; and
(3) the approximately 4770 bp BamHI-NotI fragment containing the
his gene from pING4221.
[0043] Table I, below, summarizes the content of the plasmids whose
preparation is described in Sections A through G above.
TABLE-US-00001 TABLE I Signal Plasmid BPI Product Seq. Marker 3'
Terminal Promoter pING4519 (1-199)Ala.sup.132 31AA DHFR* Human
A-MuLv Genomic HC Gamma-1 Poly-A pING4520 (1-199)Ser.sup.135 31AA
DHFR* Human A-MuLv Genomic HC Gamma-1 Poly-A pING4530
(1-199)Ala.sup.132 31AA gpt Human A-MuLv Genomic HC Gamma-1 Poly-A
pING4533 (1-199)Ala.sup.132 Kozak gpt Human A-MuLv ini- Genomic HC
tiation Seq; Gamma-1 27AA Poly-A signal pING4223 (1-193)Ala.sup.132
Kozak gpt Human A-MuLv ini- Genomic HC tiation Seq; Gamma-1 27AA
Poly-A signal pING4221 (1-193)Ala.sup.132 Kozak his Human A-MuLv
ini- Genomic HC tiation Seq; Gamma-1 27AA Poly-A signal pING4222
(1-193)Ala.sup.132 Kozak DHFR Human A-MuLv ini- Genomic HC tiation
Seq; Gamma-1 27AA Poly-A signal pING4537 (1-199)Ala.sup.132 Kozak
gpt Human A-MuLv ini- Kappa Poly- tiation Seq; A/Mouse 27AA Kappa
signal Genomic Transcription Termination pING4143
(1-193)Ala.sup.132 Kozak gpt Human A-MuLv ini- Kappa Poly- tiation
Seq; A/Mouse 27AA Kappa signal Genomic Transcription Termination
pING4146 (1-193)Ala.sup.132 Kozak DHFR Human A-MuLv ini- Kappa
Poly- tiation Seq; A/Mouse 27AA Kappa signal Genomic Transcription
Termination pING4150 (1-193)Ala.sup.132 Kozak his Human A-MuLv ini-
Kappa Poly- tiation Seq; A/Mouse 27AA Kappa signal Genomic
Transcription Termination pING4144 (1-193)Ala.sup.132 Kozak gpt
Human hCMV ini- Kappa Poly- tiation Seq; A/Mouse 27AA Kappa signal
Genomic Transcription Termination pING4145 (1-193) Kozak gpt Human
A-MuLv ini- Kappa Poly- tiation Seq; A/Mouse 27AA Kappa signal
Genomic Transcription Termination pING4148 (1-193) Kozak DHFR Human
A-MuLv ini- Kappa Poly- tiation Seq; A/Mouse 27AA Kappa signal
Genomic Transcription Termination pING4152 (1-193) Kozak his Human
A-MuLv ini- Kappa Poly- tiation Seq; A/Mouse 27AA Kappa Signal
Genomic Transcription Termination pING4151 (1-193)ala.sup.132 Kozak
his Human hCMV ini- Kappa Poly- tiation Seq; A/Mouse 27AA Kappa
signal Genomic Transcription Termination pING4154
(1-193)ala.sup.132 Kozak neo Human A-MuLv ini- Kappa Poly- tiation
Seq; A/Mouse 27AA Kappa signal Genomic Transcription Termination
*An altered DHFR gene as described in co-pending, co-owned United
States Patent Application, Ser. No. 07/885,911, incorporated herein
by reference.
EXAMPLE 2
Transfection Of Cells For Expression Of The rBPI Cysteine
Replacement Analogs
[0044] Mammalian cells are preferred hosts for production of rBPI
protein analogs according to the invention because such cells allow
for proper secretion, folding, and post-translational modification
of expressed proteins. Presently preferred mammalian host cells for
production of analogs of the invention include cells of fibroblast
and lymphoid origin, such as: CHO-K1 cells (ATCC CCL61); CHO-DG44
cells, a dihydrofolate reductase deficient [DHFR.sup.-] mutant of
CHO Toronto obtained from Dr. Lawrence Chasin, Columbia University;
CHO-DXB-11, a DHFR.sup.- mutant of CHO-K1 obtained from Dr.
Lawrence Chasin; Vero cells (ATCC CRL81); Baby Hamster Kidney (BHK)
cells (ATCC CCL10); Sp2/O-Ag14 hybridoma cells (ATCC CRL1581); and
NSO myeloma (ECACC No. 85110503).
[0045] Transfection of mammalian cells may be accomplished by a
variety of methods. A common approach involves calcium phosphate
precipitation of expression vector DNA which is subsequently taken
up by host cells. Another common approach, electroporation, causes
cells to take up DNA through membrane pores created by the
generation of a strong electric field [(Sambrook et al., Molecular
Cloning, A Laboratory Manual, Cold Spring Laboratory Harbor Press,
16.30-16.31 (1989)]. Selection for transfected cells is facilitated
by incorporation in the expression vector of a gene whose product
allows the transfected cells to survive and grow under selective
conditions. A number of such genes have been identified. These
include, among others: (1) neo, a prokaryotic gene which encodes
resistance to the aminoglycoside antibiotic G418; (2) E. coli
guanine phoshporibosyl transferase (gpt), which encodes resistance
to mycophenolic acid (MPA) in the presence of xanthine, [Mulligan
et al., Proc. Nat. Acad. Sci. USA, 78:2072-2076 (1981)]; (3)
dihydrofolate reductase (DHFR), which allows for growth of
DHFR-cells in the absence of nucleosides and gene amplification in
the presence of increasing concentration of methotrexate; (4) the
hisD gene of Salmonella typhimurium which allows growth in the
presence of histidinol [Hartman et al., Proc. Nat. Acad. Sci. USA,
85:8047-8051, (1988)]; (5) the trpB gene of E. coli [Hartman et
al., Proc. Nat. Acad. Sci. USA, 85:8047-8051, (1988)], which allows
growth in the presence of indole (without tryptophan) and (6) the
glutamine synthetase gene, which allows growth in media lacking
glutamine. The availability of these selective markers, either
alone or in various combinations, provides flexibility in the
generation of mammalian cell lines which express recombinant
products at high levels.
[0046] selection of MPA-resistant cells.
[0047] The CHO-K1 cell line is maintained in Ham's F12 medium plus
10% fetal bovine serum (FBS) supplemented with
glutamine/penicillin/streptomycin (Irvine Scientific, Irvine,
Calif.). The cells were transfected by electroporation with 40
.mu.g of pING4533 DNA which was first digested with NotI, extracted
with phenol-chloroform and ethanol precipitated. Following
electroporation, the cells were allowed to recover for 24 hours in
non-selective Ham's F12 medium. The cells were then trypsinized,
resuspended at a concentration of 5.times.10.sup.4 cells/ml in
Ham's F12 medium supplemented with MPA (25 .mu.g/ml) and xanthine
(250 .mu.g/ml) and then plated at 10.sup.4 cells/well in 96-well
plates. Untransfected CHO-K1 cells are unable to grow in this
medium due to the inhibition of pyrimidine synthesis by MPA.
[0048] At 2 weeks, colonies consisting of transfected cells were
observed in the 96-well plates. Supernatants from wells containing
single colonies were analyzed for the presence of BPI-reactive
protein by anti-BPI ELISA using rBPI(1-199) as a standard. In this
assay, Immulon-II 96-well plates (Dynatech, Chantilly, Va.) were
pre-coated with affinity purified rabbit anti-rBPI(1-199)
antiserum. Supernatant samples were added and detection was carried
out using affinity purified, biotinylated rabbit anti-rBPI(1-199)
antiserum and peroxidase-labeled avidin.
[0049] Approximately 800 colonies were screened in this manner.
Thirty-one colonies having the highest production were transferred
to 24-well plates for productivity assessment. Cells were grown to
confluence in a 24-well plate in Ham's F12 medium supplemented with
10% FBS. Once the cells reached confluence, the Ham's F12 medium
was removed and 1 ml of HB-CHO serum free medium (Irvine
Scientific) plus 40 .mu.l of sterile S-sepharose beads (Pharmacia,
Piscataway, N.J.) was added as in co-owned, co-pending U.S. patent
application Ser. No.07/885,501 by Grinna. The cells were then
incubated for 7 days after which the S-sepharose beads were removed
and washed with 0.1 M NaCl in 10 mM Tris buffer (pH7.5). The
product was eluted from the beads by addition of 1.0 M NaCl in Tris
buffer and quantitated by ELISA as described above. The
top-producing transformant, designated A153, secreted approximately
3 .mu.g/ml in this assay and was adapted to growth in Excell 301
serum-free medium (JRH Scientific, Lenexa, Kans.). The adapted
cells were grown in 1.5 L fermenters in Excell 301 medium in the
presence of S-sepharose beads. Productivity was assessed at 120-140
hours by C4 HPLC analysis of product eluted from S-sepharose beads
(50 ml aliquots). The productivity was 15-25 .mu.g/L at these
stages of the fermentation.
B. Transfection Of CHO-DG44 Cells With pING4222
[0050] Plasmid pING4222 contains DNA encoding the
rBPI(1-193)ala.sup.132 analog fused to the A-MuLv promoter,
optimized Kozak initiation sequence, human gamma-1 heavy chain 3'
untranslated region, and the mouse DHFR gene for selection of
transfected cells in a nucleoside-free medium.
[0051] The cell line, CHO DG44, was maintained in Ham's F12 medium
plus 10% FBS with glutamine/penicillin/streptomycin. The cells were
transfected with linearized pING4222 DNA (40 .mu.g digested with
PvuI, phenol-chloroform extracted, ethanol precipitated) using the
calcium phosphate method of Wigler, et al. Cell, 11:223 (1977).
Following calcium phosphate treatment, the cells were plated in
96-well plates at approximately 104 cells/well and transfectants
were obtained by growth in selective medium consisting of aMEM
medium lacking nucleosides (Irvine Scientific) and supplemented
with dialyzed FBS (100 ml serum dialyzed vs 4L cold 0.15M NaCl
using 6000-8000 MW cutoff, 16 hours, 4.degree. C.). Untransfected
CHO-DG44 cells are unable to grow in this medium due to the DHFR
mutation and the lack of nucleosides in the medium supplemented
with dialyzed serum.
[0052] At 2 weeks, each well contained approximately 2-3 colonies.
The supernatants from wells of a 96-well plate were analyzed for
the presence of rBPI(1-193) ala.sup.132 by ELISA as in Section A.
Twenty-four highest-producing clones were expanded into 24-well
plates in selective aMEM medium supplemented with 0.05 .mu.M
methotrexate to induce gene amplification of the rBPI
analog-encoding DNA. On observation of growth, cells were
transferred to a new 24-well plate and productivity was assessed
from S-sepharose eluates as described in section A for the
pING4533/CHO-K1 transfectants. The five highest-producing clones
were combined and subcloned by limiting dilution in 96-well plates.
The supernatant wells containing single colonies were assayed for
levels of rBPI(1-199) ala.sup.132 by ELISA. Twenty
highest-producing subclones were next expanded into 24-well plates
and subjected to further amplification in the presence of 0.4 .mu.M
methotrexate and the levels of product expression for the amplified
cells was determined by ELISA. The top producers, Clones 4, 75 and
80, secreted 25-37 .mu.g/ml at 7 days in a 24- well plate
containing S-sepharose.
C. Transfection Of Sp2/O Cells With pING4223 And pING4221
[0053] A strategy adopted in an attempt to achieve optimal
expression of desired rBPI products involved transfection of cells
having a first expression plasmid with a first marker, screening
for the highest producers, and then transfecting the same cells
with a second expression plasmid having a different marker. This
strategy is described below using Sp2/O cells.
[0054] Plasmid pING4223 contains DNA encoding
rBPI(1-193)ala.sup.132 BPI fused to the A-MuLv promoter, optimized
Kozak translation initiation sequence, human gamma-1 heavy chain 3'
untranslated sequences, and the gpt marker for selection of
MPA-resistant cells.
[0055] The Sp2/O cell line was maintained in DMEM medium
supplemented with 10% FBS with glutamine/penicillin/streptomycin.
The Sp2/0 cells were transfected by electroporation with 40 ug
.mu.f pING4223 DNA which had been digested with NotI and extracted
with phenol-chloroform and ethanol precipitated. Following
electroporation, the cells were allowed to recover for 48 hours in
non-selective DMEM medium. The cells were then centrifuged and
resuspended at a concentration of 5.times.10.sup.4 cells/ml in DMEM
medium supplemented with MPA (6 .mu.g/ml) and xanthine (250
.mu.g/ml) and plated at 10.sup.4 cells/well in 96-well plates.
Untransfected Sp2/O cells are unable to grow in this medium due to
the inhibition of pyrimidine synthesis by the MPA. At 1.5-2 weeks,
colonies consisting of transfected cells were observed in the
96-well plates. Supernatants from wells containing single colonies
were analyzed for the presence of product-reactive protein by
ELISA. The highest producers were transferred to a 24-well plate
and productivity was assessed in extinct 24-well cultures for cells
grown in the presence and absence of 10.sup.-7 M dexamethasone,
which causes an increase in expression by the A-MuLv promoter as a
result of interactions with the glucocorticoid receptor. The best
producer, Clone 2.times.3, secreted approximately 3 .mu.g/ml and 7
.mu.g/ml in the absence and presence of dexamethasone,
respectively.
[0056] Clone 2.times.3 was next transfected by electroporation with
pING4221, which contains the his gene for selection of
transfectants. Following recovery for 48 hours in DMEM plus 10% FBS
medium, the cells were plated in 96-well plates at approximately
10.sup.4 cells/well in DMEM/FBS supplemented with 6 .mu.g/ml MPA,
250 .mu.g/ml xanthine and 8 mM histidinol. Untransfected cells were
unable to grow in the presence of the histidinol and MPA. At 1.5-2
weeks, transfected cells were observed in the 96-well plates.
Supernatants from wells containing single colonies were analyzed
for the presence of rBPI-reactive protein by ELISA.
[0057] The highest producers were transferred to a 24-well plate.
Productivity was assessed as extinct 24-well cultures for cells
grown in the presence and absence of 10-7 M dexamethasone. The best
producer, Clone 2.times.3-130, secreted approximately 15 .mu.g/ml
and 30 .mu.g/ml in the absence and presence of dexamethasone,
respectively. This isolate was next subcloned by limiting dilution
in 96-well plates. Wells containing single colonies were screened
by ELISA and the best producers were expanded and retested in 24
well cultures in the presence and absence of 10.sup.-7 M
dexamethasone. The highest producing subclone, No. 25, secreted
approximately 16 .mu.g/ml and 33 .mu.g/ml in the absence and
presence of dexamethasone, respectively.
D. Transfection Of Sp2/0 Cells With pING4143 And pING4150
[0058] Plasmid pING4143 contains DNA encoding
rBPI(1-193)ala.sup.132 fused to the A-MuLv promoter, optimized
Kozak translation initiation sequence, and mouse kappa light chain
3' untranslated sequences along with the gpt gene for selection of
MPA-resistant cells. The Sp2/0 cells were transfected by
electroporation with 40 .mu.g of pING4143 DNA that was first
digested with NotI, phenol-chloroform extracted, and ethanol
precipitated. Following electroporation, the cells were allowed to
recover for 48 hours in non-selective DMEM medium. The cells were
then centrifuged and resuspended at a concentration of
5.times.10.sup.4 cells/ml in DMEM medium supplemented with MPA (6
.mu.g/ml) and xanthine (250 .mu.g/ml) and plated at approximately
10.sup.4 cells/well in 96-well plates.
[0059] At approximately 2 weeks, colonies consisting of transfected
cells were observed in the 96-well plates. Supernatants from wells
containing single colonies were analyzed for the presence of
BPI-reactive protein by ELISA. The highest producers were
transferred to a 24-well plate. Productivity was assessed as
extinct 24-well cultures for cells grown in the presence and
absence of 10.sup.-7 M dexamethasone. The best producer, Clone 134,
secreted approximately 12 .mu.g/ml and approximately 28 .mu.g/ml in
the absence and presence of dexamethasone, respectively.
[0060] Clone 134 was transfected by electroporation with the
vector, pING4150, which contains DNA encoding
rBPI(1-193)ala.sup.132 fused to the A-MuLv promoter and mouse light
chain 3' untranslated region with the his gene for selection of
transfectants. Prior to electroporation, the vector was first
digested, and phenol/chloroform-extracted/ethanol precipitated.
Following recovery for 48 hours in DMEM plus 10% FBS medium, the
cells were plated in 96-well plates at approximately 10.sup.4
cells/well in DMEM/FBS supplemented with 6 .mu.g/ml MPA plus 250
.mu.g/ml xanthine and 8 mM histidinol. Untransfected cells are
unable to grow in the presence of MPA and histidinol. At
approximately 2 weeks, colonies consisting of transfected cells
were observed in the 96-well plates. Supernatants from wells
containing single colonies were analyzed for the presence of
BPI-reactive protein by ELISA. The highest producers were
transferred to a 24-well plate. Productivity was assessed as
extinct 24 well cultures for cells grown in the presence and
absence of 10.sup.-7 M dexamethasone. The highest producer, Clone
134-11, was re-designated C1770. Clone C1770 secreted 36 .mu.g/ml
without dexamethasone and greater than 42 .mu.g/ml the presence of
dexamethasone. This clone (c1770) was deposited with the American
Type Culture Collection, 12301 Parklawn Drive, Rockville, Md. as
Accession No. HB 11247.
E. Transfection Of CHO-K1 Cells With pING4143
[0061] The CHO-K1 cell line was transfected with pING4143 DNA in
the manner described in Section A for transfection of CHO-K1 cells
with pING4533. At approximately 2 weeks, supernatants from
approximately 800 wells containing single colonies were analyzed
for the presence of BPI-reactive protein by ELISA. These top
producers were transferred to 24-well plates. The top producers,
secreting approximately 9-13 .mu.g/ml, may next be adapted to
serum-free medium in preparation for growth in fermenters. These
may also be re-transfected with a vector, such as pING4150 or
pING4154 with his or neo as selective markers, respectively, to
provide a cell line which produces even higher levels of rBPI
product.
F. Transfection Of CHO-K1 Cells With pING4144
[0062] Plasmid pING4144 is similar to pING4143 except that it
contains the human cytomegalovirus (hCMV) promoter instead of the
A-MuLv promoter. The CHO-K1 cell line was transfected with pING4144
DNA in the manner described above in Section A. At approximately 2
weeks, supernatants from approximately 200 wells containing single
colonies were analyzed for the presence of BPI-reactive protein by
ELISA. The top producers were transferred to 24-well plates and
rBPI expression determined in 24-well plates containing sodium
butyrate. The top producer (clone 174) secreted approximately 3-5
.mu.g/ml without butyrate and approximately 15-18 .mu.g/ml in the
presence of 5 mM butyrate in this assay. This clone, re-designated
clone C1771, was deposited with the American Type Culture
Collection, 12301 Parklawn Drive, Rockville, Md. 20852 as ATCC
accession No. CRL 11246. Top producers may next be adapted to
serum-free medium in preparation for growth in fermenters. These
may also be re-transfected with a vector, such as pING4151 or
pING4155, containing the rBPI gene under control of the hCMV
promoter, but with his or neo as selective markers, respectively,
to provide a cell line which produces even higher levels of
BPI.
G. Transfection Of NSO Cells With pING4143
[0063] NSO cells were transfected with pING4143 DNA by
electroporation. At approximately 3 weeks, colonies consisting of
transfected cells were observed in the 96-well plates. Supernatants
from wells containing single colonies were analyzed for the
presence of BPI-reactive protein by ELISA. The highest producers
were transferred to a 24-well plate. Productivity was assessed as
extinct 24-well cultures. The highest producers secreted a 15-16
.mu.g/ml. The highest producers may be retransfected with a vector,
such as pING4150, as described above to yield even higher
producers.
H. Transfection Of NSO Cells With pING4232
[0064] NSO cells were transfected by electroporation with pING4132,
which contains DNA encoding rBPI(1-193)ala.sup.132 fused to the
optimal Kozak translation initiation sequence cloned into the
vector pEE13 [Bebbington, et al. Biotechnology, 10: 169-175
(1992)]. Vector pEE13 contains the glutamine synthetase gene for
selection of transfectants which are able to grow in medium-lacking
glutamine. At approximately three weeks, colonies consisting of
transfected cells were observed in 96-well plates. Supernatants
from wells containing single colonies were analyzed by ELISA. The
highest producers were transferred to a 24-well plate. Productivity
was assessed as extinct 24-well cultures. The highest producers,
secreting 7-15 .mu.g in extinct 24 well-cultures, may next be
subjected to amplification in the presence of various
concentrations of methionine sulfoximine.
I. Transfection Of Sp2/0 Cells with pING4145
[0065] Plasmid pING4145 contains DNA encoding
rBPI(1-193)ala.sup.132 fused to the A-MuLv promoter, optimized
Kozak translation initiation sequence, mouse kappa light chain 3'
untranslated sequences, and a gpt gene for selection of
MPA-resistant cells. The Sp2/0 cells were transfected by
electroporation with 40 .mu.g of pING4145 DNA that was first
digested with NcoI, phenol-chloroform extracted, and ethanol
precipitated. Following electroporation, the cells were allowed to
recover for 48 hours in non-selective DMEM medium, centrifuged, and
resuspended at a concentration of 5.times.10.sup.4 cells/ml in DMEM
medium supplemented with MPA (6 .mu.g/ml) and xanthine (250
.mu.g/ml). The cells may then be plated at approximately 10.sup.4
cells/well in 96-well plates. At approximately 2 weeks, colonies
consisting of transfected cells are observed in the 96-well plates.
Supernatants from wells containing single colonies may then be
analyzed for the presence of BPI-reactive protein by ELISA. The
highest producers are transferred to a 24-well plate and
productivity is assessed as extinct 24-well cultures for cells
grown in the presence and absence of 10.sup.-7 M dexamethasone.
[0066] In order to maximize the expression of BPI, the highest
producing Sp2/0 transfectant may be transfected by electroporation
with a vector which contains gene sequences encoding
rBPI(1-193)ala.sup.132 fused to the A-MuLv promoter and mouse light
chain 3' untranslated region with the his gene for selection of
transfectants.
J. Transfection Of CHO-K1 Cells with pING4145
[0067] The CHO-K1 cell line was transfected with pING4145 DNA in
the manner described above in Section A. At approximately 2 weeks,
supernatants from approximately 500-800 wells containing single
colonies may be analyzed for the presence of BPI-reactive protein
by ELISA. The top producers are transferred to 24-well plates and
BPI expression determined in 24-well plates containing S-sepharose.
The top producers are next adapted to serum-free medium in
preparation for growth in fermenters. These may also be
re-transfected with a vector containing a different selective
marker to provide a cell line which produces even higher levels of
rBPI product.
K. Expression of rPBI Products from Insect Cells
[0068] Another eukaryotic system in which rBPI products may be
expressed is insect cells which have been infected with a
recombinant baculovirus containing DNA encoding an rBPI product.
Systems for generating recombinant virus and for achieving
expression of recombinant products therefrom are commercially
available (Invitrogen, San Diego, Calif.). DNA encoding
rBPI(1-199), including the 31 amino acid signal sequence, was
cloned into an NheI site in a pBlueBac transfer vector
(Invitrogen). Sf9 insect cells (BRL; ATCC CRL 1711) were
co-transfected with this vector and with wild type AcMNPV
(Autographa californica multiple nuclear polyhidrosis virus,
Invitrogen). Recombinant viral plaques were then identified,
purified, and used to generate high-titer recombinant viral stocks
as described in protocols available from BRL.
[0069] The recombinant-produced baculovirus was used to infect
further Sf9 cells. To do this, 8 separate 60 mm dishes of Sf9 cells
were infected with the baculovirus. Each of the 8 dishes was
sampled at different times during the day by collecting medium from
a dish of infected cells. Upon collection, the medium was then
centrifuged at 1000 rpm for 10 minutes and the supernatant was
stored at 4.degree. C. Cells were then rinsed once with 4 ml PBS
and lysed with 100 .mu.l/dish NP40 lysis buffer (1% NP40, 150 mM
NaCl, 50 mM Tris-HCl, pH 8.0) by incubating on ice for 30 minutes.
Cells were then collected into an Eppendorf tube with a cell
scraper. Cell lysates were then spun in a microfuge for 2 minutes.
The lysate supernatant was transferred to a new tube and stored at
-20.degree. C. Media samples from each daily time point were
analyzed for BPI content by ELISA and lysates were analyzed by
Western using an anti-BPI antibody.
[0070] No rBPI product was detectable in the media by ELISA on days
1-4 post-infection. However, on days 5-6 post-infection, a peak of
200-500 ng/ml rBPI product was detected in media samples. Western
analysis of the lysates showed a BPI-reactive band of approximately
23 Kd at day 2 post-infection. That band showed increasing
intensity through day 6.
[0071] Table II, below, summarizes the transfections detailed in
Sections A-E above. TABLE-US-00002 TABLE II Host Cell Transfected
With CHO-DG44 pING4222 CHO-K1 pING4533, pING4143, pING4144,
pING4145 NSO pING4143, pING4232 SP2/O pING4223 followed by
pING4221, pING4143 followed by pING4150, pING4145
EXAMPLE 3
[MFB--Delete this?]
Construction Of Plasmids For In Vitro Transcription And Translation
Of Rbpi (1-199)Ala.sup.132 And Rbpi (1-199)Ser.sup.135
[0072] In vitro transcription/translation studies were conducted
using plasmid pIC127 as a source of DNA encoding rBPI (1-199).
Construction of pIC127 was carried out as follows. DNA encoding
rBPI (1 - 199), including the 31 -amino acid signal sequence, was
PCR amplified from a plasmid containing full-length cDNA encoding
BPI in pGEM-72f(+). The amplification was done such that a SalI
site was incorporated at the 5' end and XhoI and SstII sites were
incorporated at the 3' end of the rBPI-encoding sequence by using
the primers BPI-3: GTCGACGCATGCGAGAGAACATGGC (SEQ ID NO: 12) and
BPI-2: CCGCGGCTCGAGCTATATTTTGGTCAT (SEQ ID NO: 9). The resulting
PCR amplified fragment was blunt-end cloned into the SmaI site of
the multiple cloning region of plasmid pT7T3 18u (Pharmacia LKB
Technology, Piscataway N.J.) in order to generate pIC102.
[0073] The pIC102 insert encoding rBPI (1 - 199) and the 31-amino
acid signal were then excised by digestion of the plasmid with
BamHI and Asp718I. A BamHI site flanks the SalI site in pIC102 and
an Asp718I site flanks the SstII site in pIC102. The ends of the
excised fragment were made blunt with T4 DNA polymerase and the
blunt fragment was then cloned into plasmid pGEM1 (Promega,
Madison, Wis.) which had first been digested with PstI and EcoRI
and blunted with T4 DNA polymerase. The resulting construction was
designated pIC124 and has the rBPI (1-199)-encoding insert oriented
such that its 5' end is adjacent to the Sp6 promoter in pGEM1.
[0074] The 31-amino acid signal sequence in the pIC124 insert was
then excised by removing the region between two HincII sites in
pIC124 to create pIC127. The excised region was replaced with a
linker which restored the initiation codon (ATG) and the sequence
encoding the first amino acid of BPI. Two fragments were isolated
from pIC124 digestion with HincII and SstII: (1) the HincII-SstII
fragment containing the rBPI (1-199) coding region excluding the
codon for the first amino acid; and (2) the SstII-HincII fragment
comprising the remainder of the plasmid. The first codon in the BPI
coding sequence and a codon for methionine in front of the BPI
sequence were inserted through use of linken formed from two
complementary annealed oligonucleotides, BPI-28: GACGCCACCATGGTC
(SEQ ID NO: 13) and BPI-29: GACCATGGTGGCGTC (SEQ ID NO: 14). Those
two oligonucleotides were ligated together with the HincII-SstII
and SstII-HincII fragments from pIC124 to form pIC127.
[0075] Two plasmids, pML101 and pML102, were constructed using
pIC127 for in vitro transcription/translation of
rBPI(1-199)ala.sup.132 and rBPI(1-199)ser.sup.135. To do this,
pIC127 was digested with SstII and EcoRI and the large SstII-EcoRI
fragment was purified. To construct pmL101, which contains an
rBPI(1-199)ala.sup.132 insert, the EcoRI-SstII fragment from
pING4519 was ligated to the SstII-EcoRI fragment form PIC127. To
construct pML102, which contains the rBPI (1-199) Ser.sup.135
insert, the EcoRI-SstII fragment from pING4520 was ligated to the
sstII-EcoRI fragment from pIC127.
[0076] BPI(1-199), rBPI (1-199) Ser.sup.135 and
BPI(1-199)ala.sup.132 were expressed in form plasmids pIC127,
pML101, and pML102 using the TNT SP6 coupled Reticulocyte Lysate
System from Pomega (Madison, Wis.). That system allows in vitro
coupled transcription and translation of cloned genes using a
eukaryotic translation system. Each coupled
transcription/translation was carried out using the manufacturer's
protocols with 2 .mu.g of plasmid DNA in a total volume of 25
.mu.l, including .sup.35S-methionine to generate labeled protein.
The labeled protein products were added in 5 .mu.l aliquots to a 20
.mu.l urea sample buffer and heated at 95.degree. C. for 3 minutes.
Aliquots (10 .mu.l) of each sample were run on a 15%
SDS-Polyacrylamide gel either with or without DTT (50 mM). After
fixing and drying the gel, the labeled protein bands were
visualized by autoradiography. Results of the autoradiography
demonstrate that cDNA encoding rBPI(1-199), rBPI(1-199)ala.sup.132,
and rBPI(1-99)cys.sup.135 expressed protein products of the
expected size of approximately 23 Kd for a BPI N-terminal fragment.
Moreover, all three expression products, rBPI(1-199),
rBPI(1-199)ala.sup.132, and rBPI(1-199)cys.sup.135, were capable of
generating higher molecular weight species of the size expected for
BPI(1-199) dimers, as well as larger species, all of which
disappeared upon reduction with DTT. It is thought that the
expression of dimeric species in the rBPI(1-199)cys.sup.135 and
rBPI(1-199)ala.sup.132 products may be the result of using a
cell-free in vitro transcription/translation system. Such a system
does not allow proper post-translational processing, folding, etc.
which would normally occur in cellular translation. Thus, it may be
that proper disulfide linkages do not always form in the in vitro
system, leading to formation of dimer in some cases.
[0077] Labeled proteins generated in the above-described in vitro
expression system were next tested for LPS binding activity. Wells
of microtiter plates were coated with LPS from Salmonella minnesota
R7 (Rd mutant) (5 mg/ml in methanol stock culture) in 0.1 M
Na.sub.2CO.sub.3/20 mM EDTA (ethylenediamine tetraacetic acid) at
pH 9.4 (a total of 2 .mu.g LPS in a 50 .mu.l well). Following
overnight incubation at 4.degree. C., the wells were rinsed with
water and dried at 37.degree. C. The wells were then blocked with
215 .mu.l Dulbecco's-PBS/0.1% BSA for 3 hours at 37.degree. C. The
blocking solution was then discarded and the wells were washed with
PBS/0.2% Tween-20. The rBPI samples were then added (2 .mu.l of the
translation reactant) to a 50 .mu.l total volume in PBS/0.2% Tween.
Following overnight incubation at 4.degree. C., the wells were
washed 3 times with PBS/0.2% Tween and the amount of labeled
protein remaining in each well was determined by liquid
scintillation counting. The results demonstrated that approximately
equivalent LPS binding took place for all three BPI species
referred to above. rBPI(1-199) displayed binding of 48,690 cpm;
rBPI(1-199)ala.sup.132 displayed binding of 59,911 cpm; and
rBPI(1-199)cys.sup.135 displayed binding of 52,537 cpm. each of the
aforementioned values represents the average of triplicate
determinations. The average binding of the control (no DNA) was
5,395 cpm.
EXAMPLE 4
Product Characterization
A. Physical Characterization
[0078] Characterization of rBPI products was accomplished using
reverse phase (C4) HPLC, cation exchange (MA7C) HPLC, SDS-PAGE, and
electrospray ionization mass spectrometry (ESI-MS). The rBPI
products to be characterized were purified from roller bottles or
from a 10 Liter fermenter harvest, by either a single-step
purification procedure or by a multi-step procedure. The
single-step procedure was essentially that disclosed in co-pending,
co-owned U.S. patent application Ser. No. 07/885,501 by Grinna,
incorporated herein by reference, with the addition of a second
wash step. In brief, S-sepharose beads were added to a growth
medium containing rBPI products. The S-sepharose was then removed
from the medium and washed with 20 mM sodium acetate and 100 mM
sodium chloride at pH4.0. A second wash was performed with 20 mM
sodium acetate and 700 mM sodium chloride at pH4.0. The purified
rBPI products were eluted with 20 mM sodium acetate and 1000 mM
sodium chloride at pH4.0.
[0079] The multi-step purification procedure involved the
purification of pooled batches of rBPI products which had first
been purified separately as described above. After purification of
each of twenty individual rBPI product batches by the single-step
method, the batches were pooled and repurified by first diluting
the salt concentration of the pooled batches to 200 mM. The pooled
sample was then loaded onto an S-sepharose column and was washed at
pH4.0 with 20 mM sodium acetate, and 200 mM sodium chloride
followed by 700 mM sodium chloride. The rBPI products were eluted
using 20 mM sodium acetate and 1000 mM sodium chloride at pH4.0.
The purified rBPI products were then analyzed to determine their
physical characteristics.
[0080] 1. SDS-PAGE Analysis of rBPI Products
[0081] SDS-PAGE analysis of rBPI products was carried out using 14%
polyacrylamide gels and a tris-glycerine buffer system under
reducing and non-reducing conditions. Protein bands were stained
with either Coomassie Blue or silver stain for visualization.
[0082] As shown in FIG. 1, non-reduced rBPI(1-199) appeared as a
major band at approximately 23 kD and a minor band at approximately
40 kD. The major band was identified as rBPI(1-199) by comparison
with simultaneously-run standards and the minor band was identified
as a dimeric form of rBPI(1-199) by immunostaining. Upon addition
of a 1/20 volume of 0.4 M dithiothreitol (DDT) to a separate sample
of rBPI(1-199), SDS-PAGE revealed a single, well-defined band
corresponding to the 23 kD monomeric species of rBPI(1-199)
identified under non-reducing conditions as described above.
[0083] SDS-PAGE analysis of the rBPI(1-199)ala.sup.132 product
revealed a single band which co-migrated with the single 23 kD
rBPI(1-199) band under reducing conditions. Under non-reducing
conditions, rBPI(1-199)ala.sup.132 migrated with the
faster-migrating of the two closely-spaced bands seen for
rBPI(1-199) (corresponding to the 23 kD band). These results, shown
in FIG. 2, indicate that rBPI(1-199)ala.sup.132 exists in
essentially monomeric form after purification. Thus, rBPI products
in which a cysteine residue is replaced by alanine display
significant resistance to dimer formation.
[0084] 2. Cation Exchange HPLC Analysis of rBPI Products
[0085] Cation exchange HPLC using an MA7C column was also employed
to measure the dimer content of rBPI products. A Bio-Rad MA7C
cartridge (4.6.times.30 mm, Bio-Rad Catalog No. 125-00556)
equilibrated with 40% buffer B (20 mM MES, 1M NaCl, pH 5.5) at 1.0
ml.min was used. The rBPI(1-199) product was analyzed by diluting a
1 ml sample to 100 .mu.g/ml and 200 .mu.l of the diluted sample was
injected onto the column. The rBPI was eluted with a gradient of
40% to 100% buffer B over 6 minutes. Buffer A comprised 20 mM MES
at pH5.5. Absorbance was monitored at 229 nm.
[0086] Analysis of rBPI(1-199) revealed two peaks. A first peak
eluted with a retention time of approximately 3 minutes as shown in
FIG. 3. A second, smaller peak eluted at approximately 6 minutes.
The first peak, shown in FIG. 3, represents rBPI(1-199) monomer and
the second peak in FIG. 3 represents rBPI(1-199) dimer as
determined by comparisons with the retention times of purified
monomer and dimer standards. The second (dimer) peak did not appear
when samples were reduced with DTT prior to being injected on the
column.
[0087] Identical procedures were used to determine the elution
pattern of rBPI(1-199)ala.sup.132. As shown in FIG. 4,
rBPI(1-199)ala.sup.132 elutes as a single peak with a retention
time corresponding to that observed for the rBPI(1-199) monomer
peak. There was no evidence of dimer in the rBPI(1-199)ala.sup.132
sample.
[0088] 3. Reverse Phase (C4) HPLC and Electrospray-Ionization Mass
Spectrometry Analysis of rBPI Products
[0089] Microheterogeneity of rBPI products was revealed by reverse
phase HPLC and electrospray-ionization mass spectrometry (ESI-MS).
For the HPLC analysis, a Brownlee BU-300 C4 column was equilibrated
with 37% Mobile Phase B (80% acetonitrile 0.065% TFA) at a flow
rate of 0.5 ml/min. Samples (1 ml each) of rBPI(1-199) were diluted
to 100 .mu.g/ml and 50 .mu.l of the sample was injected. The column
was washed with 37% Mobile Phase B for 2.5 minutes and then eluted
using a gradient from 37% to 50% Mobile Phase B over 20 minutes.
Mobile Phase A was 5% acetonitrile 0.1% TFA and absorbance was
monitored at 220 nm.
[0090] The results of reverse phase HPLC analysis of rBPI(1-199)
products are shown in FIG. 5. rBPI(l-199) products elute as a
second (major) peak with a partially-resolved first (minor) peak on
the leading edge of the second peak. Upon reduction with DTT only
one peak, corresponding to the second peak, elutes from the
column.
[0091] Identical procedures were used to analyze
rBPI(1-199)ala.sup.132 products. As shown in FIG. 6,
rBPI(1-199)ala.sup.132 eluted as a single peak corresponding to the
second (major) peak referred to above.
[0092] The eluates corresponding to the first and second HPLC peaks
described above from three separate batches of rBPI(1-199) were
isolated and analyzed to determine their content by ESI-MS.
Analysis of the eluate which produced the second (major) peak from
the rBPI(1-199) run revealed a slightly lower mass than would be
expected for a 199-amino acid protein. These data indicate that the
most abundant mass found in the second peak eluate corresponded to
a 1-193 rBPI protein fragment. However, other species, ranging in
size from 1-198 to 1-194, are also present. Analysis of the eluate
producing the single peak obtained from HPLC on
rBPI(1-199)ala.sup.132 revealed results similar to those obtained
from the eluate which produced the second (major) peak above. These
results are consistent with peptide mapping data which reveal
truncated carboxy termini in rBPI(1-199) products.
[0093] When the same analysis was performed on rBPI(1-193)
products, significantly reduced C-terminal heterogeneity was
observed. The ESI-MS data obtained from rBPI(1-193) products
revealed that approximately 85% of the protein contains either the
first 191, 193 or 193 (--an N-terminal alanine) amino acids of the
BPI N-terminal. TABLE-US-00003 TABLE III Electrospray-Ionization
Mass Spectrometry Results for rBPI(1-193) and rBPI(1-199) HPLC
Monomer Peaks Approximate Expected Approximate Predicted Amino
Relative rBPI Product Molecular Mass Acid Residues Intensity*
RBPI(1-199) 21407 1-193 50.9% 21606 1-195 28.9% 21505 1-194 20.1%
21738 1-196 <10% 21846 1-197 <10% 21966 1-198 <10%
RBPI(1-193) 21408 1-193 36.2% 21193 1-191 34.1% 21293 1-192 <10%
21477 1-193 / N- 14.5% terminal alanine 20924 1-189 15.2% *Only
species detected as being present in amounts greater than 10% were
quantitated. These species were then normalized to 100%.
[0094] These data demonstrate that, while the rBPI(1-199)-encoding
DNA produced no full-length (i.e. amino acids 1-199 of the BPI
N-terminal) protein, the rBPI (1-193)-encoding DNA produced
significant amounts of the rBPI(1-193) protein. Based upon these
and other data, it appears that significant reductions in
heterogeneity and significant increases in production of the
intended protein (i.e. that for which the DNA insert codes may be
obtained, while maintaining optimal bactericidal and LPS-binding
activity, by using truncated forms of the rBPI-encoding DNA. It is
expected that truncation of the DNA to be expressed will produce
significant reductions in heterogeneity of the expression product
to the extent that the DNA to be expressed is not truncated beyond
the cysteine at amino acid residue 175. Expression products of
truncated forms of DNA encoding rBPI proteins which have in the
range of the first 176 amino acids of the BPI N-terminal to the
first 193 amino acids of the BPI N-terminal are also expected to
retain full bactericidal and LPS-binding activity.
[0095] The ESI-MS data also revealed the presence of
microheterogeneity at the amino terminal of rBPI products. Forms of
the rBPI product having an alanine residue at the amino terminus
were found and confirmed by sequencing of tryptic peptides.
[0096] As shown in FIG. 5, the ESI-MS study of the eulate which
produced the first (minor) reverse phase HPLC peak revealed
proteins having a mass distribution similar to those which formed
the second (major) peak except that each mass value was higher by
approximately 119-120 Daltons. These data suggest that the eulate
producing the first (minor) HPLC peak described above contains a
disulfide-linked cysteine adduct, as this would account for the
uniform shift of the mass values.
[0097] To test the hypothesis that the first (minor) HPLC peak
produced by rBPI(1-199) represents cysteine adducts, rBPI(1-199)
was exposed to Ellman's reagent (dithionitrobenzenoic acid, DTNB)
which binds to free sulfhydryl groups in roughly molar equivalents.
Such treatment demonstrated that there is less than one mole of
free sulfhydryl per mole of rBPI(1-199). Given the presence of
three cysteine residues in BPI (at positions 132, 135 and 175),
these results support the notion that there is either an
intramolecular disulfide link in the rBPI products or that two of
the sulfhydryl groups are sterically unavailable.
rBPI(1-199)ala.sup.132 showed no reactivity with Ellman's
reagent.
[0098] 4. Storage Stability of rBPI(1-199) Products
[0099] Samples of rBPI(1-199) (1 mg/ml) in a buffer comprising 20
mM sodium citrate, 0.15 M Sodium Chloride buffer containing 0.1%
poloxamer and 0.002% polysorbate 80 at pH5.0 were analyzed to
determine their storage stability over an 8-week period at the
recommended storage temperature of 2- 8 C and at higher
temperatures of 22 C and 37 C.
[0100] The results for storage at 2- 8 C, presented in Table IV,
show an increase in the presence of dimer (from 1% to 4%), but no
significant increase in cysteine adduct or particle formation in
the sample. TABLE-US-00004 TABLE IV % % % LAL Storage Appearance/
Unknown Dimer Protein Cysteine Inhibition Particles/mL Particles/mL
Time Color pH Impurities by HPLC mg/mL Adduct IC.sup.50, nG/mL
.gtoreq.10 .mu.m .gtoreq.25 .mu.m initial clear/colorless 5.1 ND
1.0 1.04 12 10 230 5 4 weeks clear/colorless 5.0 ND 3.2 1.02 11 11
113 2 8 weeks clear/colorless 5.0 ND 4.4 1.02 14 9 125 5
[0101] However, storage at the increased temperatures of 22 C and
37 C show that the presence of dimer and particles in the sample
increased dramatically and the amount of cysteine adduct increased
moderately. These results are shown in Table V. Additionally, when
rBPI(1-193)ala.sup.132 is stored at 22.degree. C. to 37.degree. C.,
no dimmer was detected after storage for two weeks. Under similar
conditions, rBPI(1-199) displays significant increases.
TABLE-US-00005 TABLE V % % % LAL Storage Appearance/ Unknown Dimer
Protein Cysteine Inhibition Particles/mL Particles/mL Temperature
Time Color pH Impurities by HPLC mg/mL Adduct IC.sup.50, nG/mL
.gtoreq.10 .mu.m .gtoreq.25 .mu.m initial clear/colorless 5.1 ND
1.0 1.04 12 10 230 5 4 weeks clear/colorless 5.0 ND 4.5 1.02 12 10
100 4 22 C. 8 weeks clear/colorless 5.0 ND 6.8 1.02 14 6 126 3 4
weeks a few particles 5.0 ND 7.9 0.96 16 12 1,709 20 37 C. 8 weeks
numerous particles 5.0 ND 13.1 0.88 18 7 20,287 611
[0102] 5. Turbidity of rBPI Product Pharmaceutical Compositions
[0103] Experiments were done to determine the turbidity of various
rBPI-containing pharmaceutical compositions. In this context,
turbidity refers to the tendency of pharmaceutical compositions to
engage in unfolding (i.e., loss of tertiary protein structure)
and/or particle formation (interactions between individual proteins
to form large (>10 .mu.m) particles). The pharmaceutical
compositions tested contained either rBPI(1-199),
rBPI(1-199)ala.sup.132, or rBPI(1-193)ala.sup.132 in either a
citrate buffer (20 mM sodium citrate 150 mM sodium chloride, pH
5.0) or a citrate buffer containing 0.1% poloxamer 188 (a poloxamer
surfactant comprised of polyoxypropylene-polyoxyethylene block
copolymer) and 0.002% polysorbate 80 (a polysorbate surfactant
comprising polyoxyethylene sorbitan fatty acid ester). As mentioned
above, use of a combination poloxamer/polysorbate surfactant system
stabilizes pharmaceutical compositions as taught in co-owned,
co-pending U.S. patent application Ser. No. ______, filed on Feb.
1, 1993 [Attorney Docket No. 27129/31162] by McGregor et al.,
incorporated herein by reference.
[0104] Samples were analyzed to determine their resistance to
turbidity over time at increasing temperature and at either pH 7.0
or pH 5.0. Prior to analysis, all samples were diluted to a
concentration of 0.1 mg/ml in either 50 mM potassium phosphate or
20 mM citrate buffer at pH 7.0. Turbidity measurements were
obtained by placing samples in quartz cuvettes for use in a
Shimadzu UV-160 UV-Vis spectrophotometer (Shimadzu, Pleasanton,
Calif.) equipped with a temperature-controlled cuvette holder
attached to a recirculating water bath. Upon equilibrating the
cuvette holder at the desired temperature (57.degree. C.,
65.degree. C., or 85.degree. C., see below), absorbance at 280 nm
was measured to confirm that samples had been diluted to the proper
concentration. Following this, the absorbance of samples at 350 nm
was measured every 2 minutes for 1 hour to determine the change in
absorbance over time.
[0105] Results are presented in FIGS. 7: wherein "formulated"
refers to the rBPI product in citrate buffer containing the
poloxamer/polysorbate combination referred to above, and
"unformulated" refers to rBPI compounds in citrate buffer alone. A
lower rate of change in turbidity (i.e., a lower rate of increase
in absorbance over time) indicates increased stability against
unfolding and the formation of aggregates. A shown in FIG. 7, the
addition of the aforementioned combination of surfactants resulted
in increased stability (resistance to particle formation and
unfolding) of all compositions tested. Moreover, the
rBPI(1-199)ala.sup.132 and rBPI(1-193)ala.sup.132 exhibited greatly
improved resistance to unfolding and particle formation relative to
wild-type compositions--regardless of whether the surfactant
combination was present. Similar results were obtained at pH 5.0
and 75.degree. C. and pH 5.0 and 65.degree. C., respectively.
[0106] Overall, compositions with the surfactant combination and/or
the cysteine deletion showed greatly increased stability over time
and through increases in temperature as compared to compositions
with no surfactant and/or having the wild type BPI(1-199 N-terminal
construction.
B. In Vitro Activity Characterizations
[0107] In Vitro activity of rBPI(1-199)ala.sup.132 products was
determined by binding of the products to LPS, by the LAL inhibition
assay, and by the bactericidal potency of the products.
[0108] 1. Binding Of rBPI(1-199)ala.sup.132 To LPS
[0109] Samples (20 .mu.g to 60 .mu.g each) of E. coli (Strain
0111-B4) or S. minnesota (Rd mutant) lipopolysaccharide (Sigma
Chemical, St. Louis, Mo.) were used to determine the ability
rBPI(1-199)ala.sup.132 products to bind LPS.
[0110] The LPS samples were size fractionated by SDS-PAGE and
silver stained for visualization or electrotransferred to a
nitrocellulose membrane (BA25, Schleicher and Schuell, Keene, N.
M.) with appropriate pre-stain standards. The LPS blots were
processed by soaking the membrane in 50 mM Tris, 0.2M NaCl (TBS),
and 30 mg/ml bovine serum albumin (BSA) at pH 7.4 for 30 minutes at
37.degree. C. Membranes were then incubated in a solution
containing 2-4 .mu.g of purified or partially purified
rBPI(1-199)ala.sup.132 or a control protein (either rBPI(1-199) or
rBPI holoprotein) for 12-18 hours at 21 C to 42 C. After
incubation, the membranes were then washed with TBS-BSA. The
solution was changed at least three times over a 30 minute period.
The washed membranes were then incubated for 3 hours in a 1:1000
dilution of rabbit anti-rBPI(1-199) in TBS containing 1 mg/ml BSA.
Membranes were next washed at least three times and were developed
using the Chemiluminescent Detection System (Tropix Systems,
Bedford, Mass.) according to the manufacturers instructions, using
5.times. PBS and 0.25% gelatin (Bio-Rad) in place of I-block.
[0111] The results, shown in FIG. 11, demonstrate that
rBPI(1-199)ala.sup.132 binds to LPS fixed to nitrocellulose as well
or better than rBPI(1-199).
[0112] 2. E. coli Growth Inhibition Assay
[0113] The E. coli broth growth inhibition assay was conducted to
determine the bactericidal potency of rBPI products by treating E.
coli with rBPI(1-199) or rBPI(1-199)ala.sup.132 analogs and
monitoring the inhibition of broth growth as a measure of
bactericidal activity. A "rough" strain of E. coli with short-chain
LPS, designated J5 (a rough UDP-4-epimerase-less mutant of E.coli
strain 0111:B4), was used in the assay. Cells were grown in a
triethanolamine-buffered mineral salts medium (Simon, et al. Proc.
Nat. Acad Sci, 51:877 (1964)) which rendered E. coli especially
sensitive to BPI. The cells were washed and resuspended in 0.9%
NaCl to a density of about 5.times.10.sup.8cells/ml.
[0114] Approximately 5.times.10.sup.6 to 1.times.10.sup.7 E. coli
cells were incubated for 30-60 minutes with either rBPI(1-199) or
with rBPI(1-199)ala.sup.132 analogs at a concentration of 5
.mu.g/ml with a buffered solution (10% Hanks Balanced Salts, 40 mM
Tris-Hcl, pH 7.5. 0.1% casamino acids) in a volume of 200-400 ml.
In addition, the assay was run separately with either rBPI(1-199)
or rBPI(1-199)ala.sup.132 analog and 100 mM MgCl.sub.2. Following
incubation, the cells were diluted with 10 volumes of nutrient
broth supplemented with 0.9% Nacl. Broth growth was then monitored
for several hours.
[0115] The results, shown in FIG. 8, demonstrate that
rBPI(1-199)ala.sup.132 analogs possess bactericidal activity as
potent or more potent than rBPI(1-199). The bactericidal activity
of both rBPI(1-199)ala.sup.132 analogs and that of rBPI(1-199) were
reduced, as expected, by MgCl.sub.2.
[0116] 3. LAL Inhibition Assay
[0117] The LAL inhibition assay was used to determine the ability
of rBPI(1-193)ala.sup.132 to bind LPS. An LAL inhibition assay is
described in Gassano-Santoro, Infect. Immun., 60: 4754-4761 (1992).
Results of the LAL assay demonstrate that rBPI(1-193)ala.sup.132
has an IC-50 value of 10, which is equal to that for rBPI(1-199).
These data indicate that the analog competes as well as the
wild-type rBPI product for binding to LPS.
C. Efficacy Of rBPI(1-199)ala132 In An Animal Model Of Lethal
Endotoxemia
[0118] An animal model of endotoxemia was used to evaluate the
comparative effectiveness of rBPI(1-199)ala.sup.132 and rBPI(1-199)
against endotoxic shock.
[0119] Male ICR mice received intravenous injections of 800
.mu.g/kg actinomycin-D and either 0.5 .mu.g/kg or 1.0 .mu.g/kg of
an endotoxin (E. coli, Strain 0111:B4). Immediately following the
injection of endotoxin, the mice received an intravenous injection
(0.5 mg/kg or 5.0 mg/kg) of either rBPI(1-199) or
rBPI(1-199)ala.sup.132. Buffered vehicle was used as a control.
Deaths were then recorded over a 7-day period. The results are
presented below in Table VI. TABLE-US-00006 TABLE VI BPI Dose #
Dead/Total % Mortality Buffer only 0 14/15 93 rBPI.sub.23 0.5 mg/kg
7/15 47 5.0 mg/kg 8/15 53 rBPI.sub.23-cys 0.5 mg/kg 14/15 93 5.0
mg/kg 8/16 50
[0120] As seen in Table VI, both rBPI(1-199)ala.sup.132 and
rBPI(1-199) provided significant protection against the lethal
effects of the endotoxin. Although the present invention has been
presented in terms of its preferred embodiments, the skilled
artisan realizes that numerous modifications and substitutions are
within the scope of the present teaching. For example, substitution
of the cysteine at position 132 or 135 of the BPI N-terminal
fragment with non-polar amino acids other than alanine or serine is
contemplated by the invention. Thus, the scope of the appended
claims and any future amendments thereto.
Sequence CWU 1
1
* * * * *