Stable bactericidal/permeability-increasing protein products...

Details Stable bactericidal/permeability-increasing protein products... Stable bactericidal/permeability-increasing protein products...

2002-07-16

2004-12-07

Carlson, Karen Cochrane (Department: 1653)

Chemistry: natural resins or derivatives; peptides or proteins;

Peptides of 3 to 100 amino acid residues

C530S300000, C530S350000, C514S012200, C514S021800, C435S007100

Reexamination Certificate

active

06828418

ABSTRACT:

BACKGROUND OF THE INVENTION
This application is a continuation of U.S. application Ser. No. 09/425,034, filed Oct. 19, 1999, which is a continuation application of U.S. application Ser. No. 09/176,493, filed Oct. 21, 1998, which is a continuation of U.S. application Ser. No. 08/466,822, filed Jun. 6, 1995, now U.S. Pat. No. 5,827,816, which is a divisional of U.S. application Ser. No. 08/361,191, filed Apr. 18, 1995, now abandoned, which is a divisional of U.S. application Ser. No. 08/013,801, filed Feb. 2, 1993, now U.S. Pat. No. 5,420,019.
The present invention provides novel bactericidal/permeability-increasing protein products and stable pharmaceutical compositions containing the same.
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).
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.
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.
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 kD protein. Gazzano-Santoro et al.,
Infect. Immun
. 60:4754-4761 (1992).
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 Pat. application Ser. No. 07/885,501 by Grinna abandoned in favor of U.S. Ser. No. 08/072,063 and issued as U.S. Pat. No. 5,439,807 on Aug. 8, 1995 discloses novel methods for the purification of recombinant BPl products expressed in arid 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 BPL 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. Pat. application Ser. No. 07/885,911 by Theofan, el al. abandoned in favor of U.S. Ser. No. 08/064,693, issued as U.S. Pat. No. 5,643,570 on Jul. 1, 1997 is directed to novel, recombinant-produced BPI protcin 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.
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 as well as 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.
Even monomeric rBPI products display varying degrees of microheterogeneity in terms of the number of carboxy terminal residues present 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.
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 (polyoxypropylenepolyoxyethylene 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. Pat. application Ser. No. 08/012,360 (parent of U.S. Pat. application Ser. No. 09/190,034 which issued as U.S. Pat. No. 5,488,034) to have synergistic effects in stabilizing pharmaceutically-active polypeptides against particle format

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