Immunex Corporation

Virginia L Price

Michael C Deeley

David L Urdal

Douglas Pat Cerretti

David J Cosman

Kenneth H Grabstein

Carl J March

Thomas P Hopp

Shirley R Kronheim

Alf D Larsen

Paul J Conlon, Iii

Steven D Gimpel

Novel fusion proteins that enhance the immune response of an antigen are efficiently expressed and secreted by yeast host cells. The fusion proteins are recombinantly made by fusing the 3'-end of mature GM-CSF DNA sequence to the 5'-end of an antigen DNA sequence with or without a linker sequence. Methods of expression in yeast cells are provided.

Fusion proteins comprising GM-CSF and antigens and their expression in

A novel strain of Saccharomyces cerevisiae is useful as a host cell in the production of recombinant proteins. The novel S. cerevisiae cells transformed with a recombinant expression vector encoding a desired heterologous protein, preferably fused to a suitable N-terminal signal peptide, are cultivated under conditions that promote expression of the protein. Also provided are signal peptides derived by replacing the native signal peptidase cleavage site of a type I interleukin-1 receptor signal peptide with the tripeptide AlaXAla, wherein X represents an amino acid selected from Leu, Phe, and Gln. An expression system comprises a yeast host cell (preferably the novel S. cerevisiae strain) transformed with an expression vector comprising a promoter functional in yeast cells operably linked to DNA encoding the novel signal peptide, which is fused to the N-terminus of DNA encoding a desired heterologous protein.

Expression system comprising mutant yeast strain and expression vector

An analog human colony stimulating factor (hCSF) is disclosed, comprising a mutant amino acid sequence which is substantially homologous to the native sequence of an hCSF having at least one N-glycosylation site, wherein the mutant sequence comprises at least one amino acid substitution, deletion or insertion inactivating the N-glycosylation site.

DNA sequence encoding nonglycosylated analogs of human colony

Amplified expression of recombinant DNA products is achieved in hosts expressing proteases that cleave at multi-basic amino acid residues. To this end, cDNAs encoding granulocyte-macrophage colony stimulating factor (GM-CSF) are mutated such that one or both of the arginine residues at positions 23 and 24 of the protein product are deleted or replaced by non-basic amino acid residues. The GM-CSF analogs thus obtained maintain the activity of the wild-type protein.

Analogs of human granulocyte-macrophage colony stimulating factor

Amplified expression of recombinant DNA products is achieved in hosts expressing proteases that cleave at multi-basic amino acid residues. To this end, cDNAs encoding granulocyte-macrophage colony stimulating factor (GM-CSF) are mutated such that one or both of the arginine residues at positions 23 and 24 of the protein product are replaced by non-basic amino acid residues. The GM-CSF analogs thus obtained maintain the activity of the wild-type protein.

DNAs encoding analog GM-CSF molecules displaying resistance to proteases

Amplified expression of recombinant DNA products is achieved in hosts expressing protease that cleave at multi-basic amino acid residues. To this end, wild-type genes encoding the desired protein products are mutated by substituting codons or eliminating codons encoding multi-basic amino acid residues while maintaining the activity of the expressed protein product. Mutation of the desired gene can be conveniently carried out by site-specific in vitro mutagenisis.

Double-stranded cDNA is prepared from polyadenylated RNA extracted from activated human peripheral blood adherent mononuclear cells. The cDNA is inserted within a plasmid vector and then the recombinant plasmid employed to transform an appropriate host. Transformed hosts are identified and grouped into pools. Plasmid DNA prepared from these pools is hybridized with a labeled, synthetic oligonucleotide probe corresponding to a portion of the amino acid sequence of the interleukin 1 protein. Pools of host cells that provide a positive signal to the probe are identified, plated out and then employed in direct bacterial colony hybridization with the same probe, thereby to isolate the particular positive colony. Plasmid DNA is prepared from this colony and characterized by restriction enzyme mapping and sequencing by chain-termination method. The coding region for the IL-1 gene is inserted into a shuttle vector for amplification of the vector followed by expression of functional IL-1.

Growing community of inventors

Seattle, WA, United States of America

Virginia L Price