Recombinant production of saporin-containing proteins

Details Recombinant production of saporin-containing proteins Recombinant production of saporin-containing proteins

1995-04-13

1999-06-29

Hendricks, Keith D.

Chemistry: molecular biology and microbiology

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

Recombinant dna technique included in method of making a...

435 694, 43525233, 4353201, 536 234, C12P 2104, C12N 1562

Patent

active

059167726

DESCRIPTION:

BRIEF SUMMARY
FIELD OF THE INVENTION

The present invention relates to the recombinant production of proteins and more particularly to the recombinant production of saporin and saporin-containing fusion proteins.


BACKGROUND OF THE INVENTION

Ribosome-inactivating-proteins (RIPs) are plant proteins that catalytically inactivate eukaryotic ribosomes. RIPS have been shown to inactivate ribosomes by interfering with the protein elongation step of protein synthesis. For example, the RIP saporin (SAP) has been shown to inactivate 60S ribosomes by cleavage of the n-glycosidic bond of the adenine at position 4324 in the rat 28S ribosomal RNA (rRNA). This particular region in which A.sup.4324 is located in the rRNA is highly conserved among prokaryotes and eukaryotes. A.sup.4324 in 28S rRNA corresponds to A.sup.2660 in Escherichia coli (E. coli) 23S rRNA. Several RIP's also appear to interfere with protein synthesis in prokaryotes, such as E. coli. Since RIPs are toxic to eukaryotic cells and some RIPs are toxic to prokaryotes (see, e.g., Habuka et al. (1990) J. Biol. Chem. 265:10988-10992), they are difficult to express using recombinant DNA methodologies.
Several structurally related RIP's have been isolated from seeds and leaves of the plant Saponaria officinalis (soapwort). Among these, saporin-6 is the most active and abundant, representing 7% of total seed proteins. Saporin is very stable, has a high isoelectric point, does not contain carbohydrates, and is resistant to denaturing agents, such as SDS, and a variety of proteases. The amino acid sequences of several saporin-6 isoforms from seeds are known and there appear to be families of saporin RIPs differing in few amino acid residues.
Because saporin is a type I RIP, it does not possess a cell-binding chain, like the toxins ricin and abrin. Consequently, its toxicity to whole cells is much lower than the toxins. When targeted to cells so that it is internalized by the cells, however, its cytotoxicity is 100- to 1000-fold more potent than ricin A chain. Because of its cytotoxicity, saporin has been covalently linked to cell surface binding ligands to produce cytotoxic chemical-conjugates or linked to antibodies to produce immunotoxins that are targeted to, and internalized by, specific cells (see, e.g., Soria (1989) Pharmacological Res. 21(Supp 2):35-46, at 36). For example, basic fibroblast growth factor (bFGF) has been chemically conjugated to saporin-6 to produce the mitoxin FGF-SAP (see, e.g., U.S. Pat. No. 5,191,067 to Lappi et al.; and Lappi et al. (1989) Biochem. and Biophys. Res. Comm. 160:917-923). FGF-saporin conjugates have been used to treat restinosis (see, e.g., International Patent Application No. WO 92/11872, which is based in U.S. application Ser. No. 07/637,074). Treatment is effected by local or intravenous administration of a therapeutically effective amount of the FGF conjugate following, for example, balloon angioplasty. FGF-saporin conjugates also have shown promise as agents for the treatment of certain tumors. The growth of melanomas and other tumors that express receptors to which FGF binds can be inhibited by FGF-SAP (see, e.g., International Application No. WO 92/04918, which is based on U.S. patent application Ser. No. 07/585,319: and Beitz et al. (1992) Cancer Research 52:227-230).
An anti-human immunoglobulin heavy chain monoclonal antibody has been conjugated to saporin-6. The resulting immunotoxin is potentially useful for eliminating lymphoma and leukemia cells from human bone marrow during ex vivo treatment prior to reimplantation. Other chemical conjugates of saporin with a panel of anti-T lymphocyte monoclonal antibodies have shown promise as ex vivo agents for purging human bone marrow prior to transplantation, and as systemic therapeutic agents in patients with graft-versus-host disease and T-cell and B-cell leukemia.
Presently, conjugation of saporin to cell binding ligands and antibodies has been effected chemically. Chemical conjugation, however, results in a heterogeneous population of molecules. For example, bFGF is conjugate

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