Therapeutic peptide-based constructs derived from domain II...

Details Therapeutic peptide-based constructs derived from domain II... Therapeutic peptide-based constructs derived from domain II...

1999-06-25

2003-02-04

Russel, Jeffrey E. (Department: 1653)

Chemistry: natural resins or derivatives; peptides or proteins;

Peptides of 3 to 100 amino acid residues

11 to 14 amino acid residues in defined sequence

C514S014800, C514S015800, C514S016700, C530S326000, C530S328000

Reexamination Certificate

active

06515104

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates generally to small peptide-based constructs that have 8 to 15 amino acid moieties. The sequences of these peptide-based constructs are designed and prepared based on a reverse subsequence (99-85) derived from amino acids identified and selected from Domain II of bactericidal/permeability-increasing protein (BPI). The invention further relates to therapeutic uses of such peptide-based constructs due to their properties of heparin binding and neutralization, inhibition of endothelial cell proliferation and/or inhibition of angiogenesis, e.g., inhibition of in vivo neovascularization, including in models of chronic inflammatory disease states and metastatic tumors.
BACKGROUND OF THE INVENTION
Bactericidal/permeability-increasing protein (BPI) is a protein isolated from the granules of mammalian polymorphonuclear neutrophils (PMNs), which are blood cells essential in defending a mammal against invading microorganisms. Human BPI has been isolated from PMNs by acid extraction combined with either ion exchange chromatography (Elsbach, 1979,
J. Biol. Chem.
25: 11000) or
E. coli
affinity chromatography (Weiss et al., 1987,
Blood
69: 652), and has bactericidal activity against gram-negative bacteria. The molecular weight of human BPI is approximately 55,000 daltons (55 kD). The amino acid sequence of the entire human BPI protein and the nucleic acid sequence of DNA encoding BPI, have been reported by Gray et al., 1989,
J. Biol. Chem.
264: 9505 (see FIG. 1 in Gray et al.). The Gray et al. DNA and amino acid sequences are set out in SEQ ID NOS: 12 and 13 hereto.
The bactericidal effect of BPI was originally reported to be highly specific to sensitive gram-negative species. The precise mechanism by which BPI kills gram-negative bacteria is not yet known, but it is known that BPI must first attach to the surface of susceptible gram-negative bacteria. This initial binding of BPI to the bacteria involves electrostatic interactions between BPI, which is a basic (ie., positively charged) protein, and negatively charged sites on lipopolysaccharides (LPS). LPS is also known as “endotoxin” because of the potent inflammatory response that it stimulates. LPS induces the release of mediators by host inflammatory cells which may ultimately result in irreversible endotoxic shock. BPI binds to Lipid A, the most toxic and most biologically active component of LPS.
BPI is also capable of neutralizing the endotoxic properties of LPS to which it binds. Because of its gram-negative bactericidal properties and its ability to bind to and neutralize LPS, BPI can be utilized for the treatment of mammals suffering from diseases and conditions initiated by infection with gram-negative bacteria whether the bacteria infect from outside the host or the bacteria infect from within the host (i.e., gut-derived), including conditions of bacteremia, endotoxemia, and sepsis. These properties of BPI make BPI particularly useful and advantageous for such therapeutic administration.
A proteolytic fragment corresponding to the amino-terminal portion of human BPI possesses the LPS binding and neutralizing activities and antibacterial activity of BPI holoprotein. In contrast to the amino-terminal portion, the carboxyl-terminal region of isolated human BPI displays only slightly detectable antibacterial activity and some endotoxin neutralizing activity (Ooi et al., 1991,
J. Exp. Med.
174: 649). One BPI amino-terminal fragment, referred to as “rBPI
23
” (see Gazzano-Santoro et al., 1992,
Infect. Immun.
60: 4754-4761) has been produced by recombinant means as a 23 kD protein and comprises an expression product of DNA encoding the first 199 amino acid residues of the human BPI holoprotein taken from Gray et al., supra, except that valine at position 151 is specified by GTG rather than GTC and residue 185 is glutamic acid (specified by GAG) rather than lysine (specified by AAG). Recombinant holoprotein, also referred to as rBPI, has also been produced having the sequence set out in SEQ ID NOS: 12 and 13 taken from Gray et al., supra, with the exceptions noted for rBPI
23
. An N-terminal fragment analog designated rBPI
21
or rBPI
2
&Dgr;cys or rBPI (1-193) ala
132
has been described in co-owned U.S. Pat. No. 5,420,019 and corresponding International Publication No. WO 94/18323 (PCT/US94/01235). This analog comprises the first 193 amino acids of BPI holoprotein as set out in SEQ ID NOS: 12 and 13 but wherein the cysteine at residue number 132 is substituted with alanine, and with the exceptions noted for rBPI
23
. rBPI
23
, as well as the cysteine substitution analog designated rBPI
21
, have been introduced into human clinical trials. Proinflammatory responses to endotoxin were significantly ameliorated when rBPI
23
was administered in humans challenged with endotoxin. (See, e.g., co-owned U.S. Pat. Nos. 5,643,875 and 5,753,620 and corresponding International Publication No. WO 95/19784 (PCT/US95/01151). In addition, rBPI
21
was administered in humans with meningococcemia and hemorrhage due to trauma. (See, e.g., U.S. Pate. No. 5,888,977 and corresponding International Publication No. WO 97/42966 (PCT/US97/08016) and U.S. Pat. No. 5,756,464 and corresponding International Publication No. WO 97/44056 (PCT/US97/08941).
Other endotoxin binding and neutralizing proteins and peptides are known in the art. One example is Limulus antilipopolysaccharide factor (LALF) from horseshoe crab amebocytes (Warren et al., 1992,
Infect. Immunol.
60: 2506-2513). Another example is a cyclic, cationic lipopeptide from
Bacillus polymyxa,
termed Polymyxin B
1
. Polymyxin B
1
is composed of six &agr;,&ggr;-diaminobutyric acid residues, one D-phenylalanine, one leucine, one threonine and a 6-methyloctanoyl moiety (Morrison and Jacobs, 1976,
Immunochem.
13: 813-818) and is also bactericidal. Polymyxin analogues lacking the fatty acid moiety are also known, which analogues retain LPS binding capacity but are without appreciable bactericidal activity (Danner et al., 1989,
Antimicrob. Agents Chemother.
33: 1428-1434). Similar properties have also been found with synthetic cyclized polymyxin analogues (Rustici et al., 1993, Science 259: 361-365).
Known antibacterial peptides include cecropins and magainins. The cecropins are a family of antibacterial peptides found in the hemolymph of lepidopteran insects (Wade et al., 1990,
Proc. Natl. Acad. Sci. USA
87: 4761-4765), and the magainins are a family of antibacterial peptides found in Xenopus skin and gastric mucosa (Zasloffet al., 1988,
Proc. Natl. Acad. Sci. USA
85: 910-913). These peptides are linear and range from about 20 to about 40 amino acids in length. A less active mammalian cecropin has been reported from porcine intestinal mucosa, cecropin P1 (Boman et al., 1993,
Infect. Immun.
61: 2978-2984). The cecropins are generally reported to be more potent than the magainins in bactericidal activity and appear to have less mammalian cell cytotoxicity. The cecropins and magainins are characterized by a continuous, amphipathic &agr;-helical region which is necessary for bactericidal activity. The most potent of the cecropins identified to date is cecropin A. The sequence of the first ten amino acids of the cecropin A has some homology with the BPI amino acid sequence 90-99 but does not share the motif of charged and uncharged amino acids specified by the BPI amino acid sequence 90-99. In addition, the other 27 amino acids of cecropin A are necessary for maximal bactericidal activity and there is no homology with BPI for those 27 amino acids. The magainins have minimal homology with the BPI amino acid sequence 90-99.
Of interest to the present application are the disclosures in PCT International Application PCT/US91/05758 [WO 92/03535] relating to compositions comprising BPI and an anionic compound, which compositions are said to exhibit (1) no bactericidal activity and (2) endotoxin neutralizing activity. Anionic compounds are preferably a protein such as serum albumin but can also be a polysaccharide such as heparin. In a

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