Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai
Reexamination Certificate
1996-11-14
2001-10-16
Priebe, Scott D. (Department: 1632)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Peptide containing doai
C530S310000, C530S324000, C530S326000
Reexamination Certificate
active
06303568
ABSTRACT:
BACKGROUND OF THE INVENTION
This invention relates to certain therapeutic polypeptides useful in man and animals. Specifically, the therapeutic polypeptides are useful in cases of intra- and extracellular bacteria, fungi and protozoa which are either resistant to conventional antibiotics, difficult to treat without harm to the host cells, associated with severe infections, or connected with cases of traumatized or immune compromised hosts. In view of the properties of the therapeutic polypeptides of this invention which include definite antibacterial, anti-fungal and anti-protozoan activity, the therapeutic polypeptides provided herein are termed antimicrobial polypeptides.
Further, this invention relates to biosynthetic processes affording the antimicrobial polypeptides, including cloning or producing the desired polypeptides in various media, both as active antimicrobial polypeptides and also in the pro-form or the inactive antimicrobial polypeptide form with subsequent activation procedures. Included in these processes and procedures for biosynthesis of the antimicrobial polypeptides of this invention are novel forms of the polypeptides themselves, synthetic or semi-synthetic polypeptides and cells, containing such novel polypeptides which are themselves novel compositions, or cell lines, or hybridoma.
Still further, this invention relates to novel cells, including animal cells, and particularly mammalian cells, containing such antimicrobial polypeptides, including various specific mammalian cell types which contain the antimicrobial polypeptides. Methods for treating mammals infected with certain bacteria, protozoa, or fungi, and which are resistant to known antibiotics or which are difficult to treat with such antibiotics are also included in the present invention. As a novel composition, the present invention includes specifically mammalian cells having genes encoding for such antimicrobial polypeptides; particularly, various specific embryonic cells having the genes encoding for the antimicrobial polypeptides are included in the present invention. Additionally, processes for and methods of preparing mammalian cells including the genes encoding for antimicrobial polypeptides of this invention using recombinant DNA techniques are a part of the present invention.
A number of the antimicrobial polypeptides have been found to be useful when the genes encoding therefor are incorporated into various plant species. Particularly, when introduced into the plant genome by means of Agrobacterium, the antimicrobial polypeptide encoding genes produce plant species much more resistant to certain bacterially induced disease conditions and plant pathogens. Such antimicrobial polypeptides and the incorporation of the genes encoding therefor are more fully described in U.S. patent application Ser. No. 889,225, filed Jul. 25, 1986, to Jaynes et al, which is incorporated herein by reference as if fully set forth.
Although antimicrobial polypeptides of the type envisioned in the present invention are known from the humoral response to bacterial infection of the
Hyalophora cecropia
(a species of large silk moth), prior to the discovery of Jaynes et al, supra, it was unknown that such antimicrobial polypeptides were useful against plant pathogens or how the same could be transformed into plants. It is likewise previously unknown before the present invention, that the antimicrobial polypeptides would be similarly effective in animal, and particularly mammalian species, against certain microorganisms, or what method could be employed to transform cells of such species therewith.
It is known that certain polynucleotide molecules can be expressible in a given host and have the sequence araB promoter operably linked to a gene which is heterologous to such host. The heterologous gene codes for a biologically active polypeptide. A genetic construct of a first genetic sequence coding for cecropin operably linked to a second genetic sequence coding for a polypeptide which is capable of supressing the biological effect of the resulting fusion protein towards an otherwise cecropin-sensitive bacterium, International Patent Publication W086/04356, Jul. 31, 1986.
It is therefore an object of the present invention to provide antimicrobial polypeptides for therapeutic treatment of pathogens in mammals including bacteria, fungi, and protozoa. A further object is to provide a method for treatment of man and animals having a bacterial infection with an antimicrobial polypeptide for such infection. A still further object of the present invention is to provide for a biosynthetic process to produce such antimicrobial polypeptides. Another object of the present invention is to provide for novel synthetic and semisynthetic antimicrobial polypeptides produced by recombinant DNA procedures. A still further object of the invention provides cells from a host animal transformed by genetic sequences incorporated into the cells, and ultimately to provide transformed animals which are as a result resistant to a number of pathogenic microorganisms.
These and still other objects of the invention are provided according to the present invention as described in the following specification.
THE INVENTION
The present invention provides for a method for treatment of an animal having a bacterial infection caused by either or both gram-positive or gram-negative bacteria, such as by a member of the group consisting of Brucella, Listeria, Pseudomonas (other than
P. solanacium
), Staphylococcus or a protozoan infection caused by a member of the group consisting of Trypanosoma and Plasmodia, which method comprises administration to said mammal of an antibacterial amount of an antimicrobial polypeptide selected from the group consisting of a cecropin, an attacin, a lysozyme, a polypeptide transcribed from gene 13 of phage P22, an S protein from lambda phage, and an E protein from phage PhiX174. As another aspect of the present invention, there is provided a biosynthetic method for producing the antimicrobial polypeptides of the present invention which method includes the steps of
(a) microinjecting a phosphate buffered solution of genes encoding for and an antimicrobial polypeptide into selected cells, and
(b) culturing the cells to produce such antimicrobial polypeptide included in the genome of said cells.
In a still further aspect, precursor polypeptides for the antimicrobial polypeptides of the present invention are produced by a process which comprises the steps of:
(a) augmenting at the 5′ end in the correct reading frame the genes encoding for the beta-lactamase gene of
E. coli
by fusing thereto genes encoding for at least one antimicrobial polypeptide free from internal methionine codons and having a methionine codon at the 5′ end of the antimicrobial gene to produce an augmented
E. coli;
(b) culturing the augmented
E. coli
under conditions to produce beta-lactamase modified by containing at least one said antimicrobial polypeptide;
(c) isolating the modified beta-lactamase;
(d) treating the modified beta-lactamase with cyanogen bromide to cleave the antimicrobial polypeptide; and
(e) separating the antimicrobial polypeptide from the beta-lactamase, whereby an active antimicrobial polypeptide is formed.
The antimicrobial polypeptides of the present invention are relatively small having from about 30 to about 40 amino acids and, generally, as produced in natural settings include longer chains of amino acids, for example, of up to about 160 units in length. However, it is believed that the active portion of the naturally produced material selected for antimicrobial activity is between about 30 and about 40 amino acid units in length. Such a length is unusually small to have a widespread effect as an antimicrobial polypeptide. However, the in vitro tests, discussed below, have shown such antimicrobial activity to be present. Antimicrobial effects in insects have been noted for certain of the antimicrobial polypeptides. Specifically, the cecropins, attacins, and lysozymes were determined to be present in the humor
Enright Frederick M.
Jaynes Jesse M.
White Kenneth L.
Baker & Botts L.L.P.
Beckerleg Anne Marie S.
Helix Biomedix, Inc.
Priebe Scott D.
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