New patent, issued today 13 Aug.
SUMMARY OF THE INVENTION
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.
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 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 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 codon for leucine at position 193.
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 automonously-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 that 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.
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.
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).
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. |
