Methods for the preparation of positively charged proteins

Details Methods for the preparation of positively charged proteins Methods for the preparation of positively charged proteins

1997-06-30

2001-08-14

Chan, Christina Y. (Department: 1644)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Using a micro-organism to make a protein or polypeptide

C435S383000, C435S395000, C435S404000, C530S350000

Reexamination Certificate

active

06274348

ABSTRACT:

FILED OF THE INVENTION
The present invention generally relates to recombinant hybrid fusion proteins useful in treating bacterial infection in humans, DNA sequences encoding such proteins, recombinant methods for preparing the proteins, and pharmaceutical preparations containing the recombinant products. Hybrid fusion proteins of the present invention are expression products of direct transcriptional fusions of DNAs encoding bactericidal/permeability-increasing protein or a biologically active fragment thereof and DNAs encoding one or more immunoglobulin heavy chain constant regions, which fusions have been incorporated into a suitable plasmid vector and transfected or transformed into a host cell. The recombinant-produced BPI-Immunoglobulin fusion protein expression products (hereinafter “rBPI-Ig”) are useful as endotoxin binding proteins and as bactericidal agents.
BACKGROUND OF THE INVENTION
Bactericidal/permeability-increasing protein (hereinafter “BPI”) is a cationic protein which binds to the lipid A portion of bacterial lipopolysaccharide (hereinafter “LPS”). Binding of the BPI protein to bacterial LPS increases the envelope permeability of susceptible gram negative bacteria. Ooi, et al.,
J. Biol. Chem
., 262:14891 (1987). BPI also binds to soluble LPS. Human BPI protein has been isolated from polymorphonuclear neutrophils (hereinafter “PMNs”) by acid extraction combined with either ion exchange chromatography or
E. coli
affinity chromatography. Elsbach, et al.
J. Biol. Chem
., 254:11000 (1979); Weiss et al.,
Blood
, 69:652 (1987).
The holo-BPI protein isolated from human PMNs has potent bactericidal activity against a broad spectrum of gram-negative bacteria. Elsbach, et al.,
J. Biol. Chem
., 254:11000 (1979). This antibacterial activity appears to be associated with the amino terminal region of the isolated human holo-BPI protein. In contrast, 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). Human DNA encoding BPI has been cloned and the amino acid sequence of the encoded protein has been elucidated. Gray et al.,
J. Biol. Chem
., 264:9505-9509 (1989); U.S. Pat. No. 5,198,541.
Immunoglobulins comprise a family of proteins with numerous structural similarities, but also important structural differences which result in differences in antigen binding properties and other biological activities. For example, the IgG isotype antibodies have the longest serum half life and are susceptible to placental transfer. The most potent anti-viral activity is associated with the IgA isotype antibodies; while the IgM isotype antibodies possess the greatest antibacterial efficacy. Stites, et al.
Basic and Clinical Immunology
, p. 32 (Appleton & Lange, 6th ed. 1987). Within each isotype of antibody in the immunoglobulin family there exist several subclasses and allotypic variations. Id.
Members of the so-called “immunoglobulin gene superfamily” generally possess extracellular domains which are characterized by multiple looping due to the formation of disulfide bridges. Such looping occurs in the heavy chain constant domains, (designated CH1, CH2, CH3, and CH4) of immunoglobulin molecules. Non-immunoglobulin compounds possessing multiple looping domains which are homologous to those present in members of the immunoglobulin superfamily also exist and certain of these have been termed “adhesons”. See, e.g., PCT Application No. WO89102922, published Apr. 6, 1989; Capon et al.,
Nature
, 337:525-531 (1989).
Of particular interest to the present invention are reports of the recombinant synthesis of hybrid fusion proteins involving a portion of an adheson as the first component and an immunoglobulin heavy chain constant region as a second component of the fusion. See, e.g., Harris,
Eur. J. Biol. Chem
., 194:611-620 (1990) and Capon et al., supra, addressing the formation of CD4/IgG fusions. The structural design rationale for such a molecule is based on the observation that the adheson component of the fusion has a structure similar to that of the immunoglobulin component, and would therefore be expected to fold in a manner which is complementary to that of the immunoglobulin component. See Gascoigne, et al.,
P.N.A.S
. (
USA
), 84:2936-2940 (1987) addressing recombinant chimeric T-cell receptor-immunoglobulin proteins. See also Mariuzza, et al.,
J. Biol. Chem
., 264(13):7310-7316 (1989); Goverman, et al.,
Cell
60:929-939 (1990); Gregoine, et al.
P.N.A.S
. (
USA
), 88:8077-8081 (1991); Bismuth, et al.,
Molecular Inmmunol
., 27(11):1127-1136(1990) (addressing similar T-cell receptor-immunoglobulin fusions). A soluble CD44-immunoglobulin fusion protein has also been reported. Aruffo, et al.,
Cell
61:1303-1313 (1990).
Ashkenazi, et al.,
P.N.A.S
. (
USA
), 88:1035 (1991) report protection against endotoxic shock through use of a chimeric immuno-adhesin protein (an adheson variant) which acts as a tumor necrosis factor (TNF) antagonist. The TNF antagonist reported therein is a hybrid fusion protein wherein the extracellular portion of a TNF receptor (TNFR) protein is fused to a constant domain of a human IgG heavy chain. This TNFR-IgG fusion reportedly binds to and blocks the cytotoxic effect of TNF on actinomycin-D treated cells and provides protection against endotoxin challenge when administered prior to the endotoxin.
Significantly, all of the fusion proteins described above have involved molecules that are expressed on the surface of cells as integral membrane proteins and, with the exception of CD44 and the TNF receptor, have possessed structures characteristic of the immunoglobulin gene superfamily.
Published PCT application WO92/03535 reports the construction of fusion of an amino-terminal portion of BPI to cDNA encoding the constant domain of IgG. However, that report fails to indicate how such protein-cDNA constructions are made and further fails to teach how many other types of BPI-Ig fusion may be constructed.
SUMMARY OF THE INVENTION
The present invention provides novel hybrid fusion proteins useful in treating bacterial infection and the sequelae thereof. Also provided are DNA sequences encoding such proteins, recombinant methods for preparing the proteins, and pharmaceutical preparations containing the recombinant products.
According to one aspect of the invention, hybrid fusion proteins are provided which, at their amino terminal, comprise a bactericidal/permeability-increasing protein or a biologically active fragment thereof fused to at least one constant domain of an immunoglobulin heavy chain or an allelic variant thereof which forms the carboxy terminal of the fusion protein.
In a preferred embodiment of the invention, the immunoglobulin heavy chain constant region portion of the fusion comprises two domains of the heavy chain constant region and most preferably the CH2 and CH3 domains. Fusion proteins of the present invention may also possess an immunoglobulin hinge region between the immunoglobulin and BPI regions.
Immunoglobulin heavy chain constant domains useful in formation of hybrid proteins of the present invention may be patterned after any isotype, but are preferably based on IgG, IgA, or IgM isotypes or allelic variants of those isotypes.
In presently preferred embodiments of the invention, hybrid fusion proteins comprise an amino terminal fragment including from 176 to 199 of the initial amino terminal residues of the mature human BPI protein. Also, the BPI portion of the fusion may comprise a BPI analog, wherein the cysteine residues at either or both of positions 132 and 135 of the native BPI sequence is replaced by another amino acid, preferably alanine or serine. When produced by recombinant methods, fusion proteins are isolated in monomeric or homodimeric forms.
According to another aspect of the invention, DNA sequences are provided which encode the above-described hybrid fusion proteins. Also provided are autonomously replicating DNA plasmid vectors including such DNA sequences and host cells stably transformed or transfected with suc

Affiliated with

Also associated with

Rating

Methods for the preparation of positively charged proteins does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Methods for the preparation of positively charged proteins, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Methods for the preparation of positively charged proteins will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-2464280